Demarcating the gene-rich regions of the wheatgenomeMustafa Erayman Devinder Sandhu Deepak Sidhu Muharrem Dilbirligi

P S Baenziger1 and Kulvinder S Gill

Washington State University Pullman WA 99164 USA and 1Department of Agronomy and HorticultureUniversity of Nebraska-Lincoln Lincoln NE 68583-0915 USA

Received January 14 2004 Revised March 28 2004 Accepted May 14 2004

ABSTRACT

By physically mapping 3025 loci including 252phenotypically characterized genes and 17 quan-titative trait loci (QTLs) relative to 334 deletionbreakpoints we localized the gene-containing frac-tion to 29 of the wheat genome present as 18major and 30 minor gene-rich regions (GRRs) TheGRRs varied both in gene number and density Thefive largest GRRs physically spanning 3 of thegenome contained 26 of the wheat genesApproximate size of the GRRs ranged from 3 to 71Mb Recombination mainly occurred in the GRRsVarious GRRs varied as much as 128-fold for genedensity and 140-fold for recombination ratesExcept for a general suppression in 25ndash40 of thechromosomal region around centromeres nocorrelation of recombination was observed with thegene density the size or chromosomal locationof GRRs More than 30 of the wheat genes are inrecombination-poor regions thus are inaccessibleto map-based cloning

INTRODUCTION

Bread wheat (Triticum aestivum L) is an allohexaploid(2n = 6x = 42 AABBDD) containing three homoeologousgenomes (1) Wheat genome is about 35 times larger thanrice (Oryza sativa L) although both belong to Poaceaefamily (2) Estimates for the gene-containing fraction of thewheat genome range from 1ndash5 obtained from the availablesequence data analyses and genome size comparisons withother plant genomes to 15 by DNA re-association kineticsexperiments (3ndash5) It is therefore imperative to identify anddemarcate the gene-containing regions for an efficient andtargeted characterization of this important genome Second

genetic maps and DNA sequence comparisons along withother phylogenetic studies have suggested that various grassgenomes originated from a common ancestor (67) This is alsoevident from the fact that majority of wheat barley (Hordeumvulgare L) and rice protein sequences are 98 similar (8) Itis however unknown how monophyletic origin of the grassesresulted in as much as 35-fold difference in genome size

Genes are unevenly distributed on wheat as well as otherplant chromosomes (59ndash12) In Arabidopsis thaliana 45of the genome accounts for all 25 000 genes (513) Theremaining 55 is lsquogene-emptyrsquo and is interspersed amonggenes as blocks ranging in size from a few hundred basepairsto 50 kb Gene-rich and gene-poor regions were alsoobserved in pea tomato and palm (14) Uneven distributionof genes on chromosomes seems to be a common feature ofother higher eukaryotes also (15ndash17) In larger genomessuch as human unevenness of gene distribution is morepronounced (18) The 30 000 genes with an average size of27 kb account for 25 of the human genome The remaining75 is composed of retrotransposon-like repetitive sequencesinterspersed among genes as a result of multiple invasionsby different retrotransposons at different times duringevolution followed by their inactivation by transpositionandor heterochromatinization (18ndash20)

By physically mapping gene markers on an array of chromo-some deletion lines it has been shown that most wheat genesare present in clusters that occur more frequently in distal partsof the chromosomes (92122) The exact location relativesize gene density and structural organization of the gene-containing regions are however not known Comparisons ofgenetic distances among C-bands revealed that distribution ofrecombination is also uneven along the wheat chromosomes(2324) Comparisons of wheat physical and genetic linkagemaps confirmed the uneven distribution of both genes andrecombination and established a general correlation betweenthe two (9102125ndash29) Precise relationships among rate ofrecombination distribution of genes gene density and locationon the chromosome have not been established in wheat

To whom correspondence should be addressed at Department of Crop and Soil Sciences 277 Johnson Hall PO Box 646420 Washington State UniversityPullman WA 99164 USA Tel +1 509 335 4666 Fax +1 509 335 8674 Email ksgillwsueduPresent addressesDevinder Sandhu G302 Agronomy Hall Department of Agronomy Iowa State University Ames IA-50011-1010 USAMustafa Erayman Agricultural College Department of Crop Sciences Mustafa Kemal University 31034 Hatay TurkeyMuharrem Dilbirligi Central Research Institute for Field Crops Eskisehir yolu 10 km LodumuluAnkara Pk 226 0642 UlusAnkara Turkey

The authors wish it to be known that in their opinion the first four authors should be regarded as joint First Authors

Nucleic Acids Research Vol 32 No 12 ordf Oxford University Press 2004 all rights reserved

3546ndash3565 Nucleic Acids Research 2004 Vol 32 No 12doi101093nargkh639

Published online July 7 2004

Regions around the eukaryotic centromeres and in some casestelomeres have been reported to suppress recombinationrates (133031) Furthermore as shown in barley maize(Zea mays L) Arabidopsis and other plants distribution ofrecombination can be highly uneven even within a few kilobases of DNA (32ndash35) Relationships of the similar putativerecombination hotspots of wheat with the currently identifiedhighly recombinogenic chromosomal regions have not beenestablished

About 461 phenotypic markers and useful genes have beenidentified in wheat (36) (httpwheatpwusdagov) Althoughthe linkage relationship of about 377 has been establishedrelative to molecular markers the physical location of onlyseven of these genes is known (37) Because of uneven dist-ribution of recombination the physical location of the genesalong with a precise kbcM estimate for the region areessential for map-based cloning This is particularly importantfor wheat where the difference in the recombination amongregions is expected to be greater than other crop plants

The objectives of this study were to generate a comprehens-ive map of the wheat genome identify and precisely localizethe gene-containing regions reveal gene density distributionof recombination and kbcM estimate for each of thegene-containing regions and physically map phenotypicallycharacterized genes

MATERIALS AND METHODS

Plant material

Various aneuploid stocks along with the wild-type wheat culti-var Chinese Spring (CS) were used for physical mapping The21 nullisomicndashtetrasomic lines (NT missing a pair of chromo-somes the deficiency of which is compensated for by an extrapair of homoeologous chromosomes) were used for inter-chromosomal mapping and 14 ditelosomic lines (DT missinga pair of chromosome arms) were used to reveal arm location ofthe gene markers Sub-arm localization of markers was accom-plished using 334 deletion lines covering all 21 wheat chromo-somes A list of the deletion lines along with the fraction length(FL) of the retained arm is provided as supplementary informa-tion in Supplementary Table 1 These deletion lines weregenerated using gametocidal genes from Aegilops speltoides(38ndash40) Giemsa C-banding characterization and low-densityrestriction fragment length polymorphism (RFLP) mappingsuggested that most of these deletion lines resulted from singlebreaks followed by the loss of the acentric fragments (262740)High-density mapping revealed a few complex and secondarydeletionsrearrangements in some of the deletion lines (10)Additional information about the deletion lines is available athttpwwwksueduwgrcGermplasmDeletions

DNA analysis and marker selection

Plant genomic DNA was extracted following a previouslydescribed method (41) Gel blot analysis was performedusing 15 g of genomic DNA digested with either EcoRI orHindIII restriction enzymes and size separated on 08 agar-ose gels All other steps for the gel blot DNA analysis were aspreviously described (26)

The published and the Poaceae maps from the lsquograingenesrsquodatabase (httpwheatpwusdagovggpagesmapshtml) wereused to select gene markers for physical mapping The DNAmarkers were from wheat (FBA FBB NOR PSR TAGTAM UNL WG) Ae tauschii (KSU) barley (ABC ABGBCD MWG and cMWG) oat (CDO) and rice (RZ) (104243)Priority was given to cDNA clones although some PstI geno-mic clones were also used

Deletion mapping

Chromosomal and arm locations of each fragment band wererevealed by the NT and DT mapping A fragment band wasmapped to a chromosomal region bracketed by the breakpointsof the largest deletion possessing the fragment band and thesmallest deletion lacking it Multiple fragments were consid-ered non-allelic (shown by a letter at the end of the probename) if the corresponding bands mapped to different regionsor if more than two DNA fragments larger than the probe sizewere detected

Demarcating the gene-rich regions

The deletion mapping information for the three homoeologs ofeach group was combined to generate a high-resolution con-sensus physical map All deletion breakpoints for a group werealigned on a hypothetical chromosome based on FL values andeach marker was placed in the shortest possible interval basedon its location on the three homoeologs Due to the occasionaldifferences in size and distribution of gene-poor regionsamong homoeologs relative locations of markers were alsoconsidered along with the FL values to place deletion break-points on the consensus map To determine physical locationof a GRR the FL value consistent with at least two of thethree homoeologs was used Deletion mapping data fromthe three homoeologs along with FL location were used tolocalize deletion breakpoints within a GRR

Recombination analysis

A comprehensive genetic linkage map for each of the sevenwheat homoeologous groups was constructed using 137 geneticlinkage maps including 17 each for group 1 and 2 32 for 3 25 for4 16 for 5 and 15 each for group 6 and 7 chromosomes (httpwheatpwusdagovggpagesmapsshtml) First the markersgenes that were common among several wheat genetic linkagemaps for a homoeologous group were designated as anchorsTotal genetic length of the consensus map and distances amonganchors were estimated by taking average of recombinationamong anchor markers from various maps Second markerspresent between two anchor markers on multiple maps wereintegrated on the consensus map Relative genetic distancesof the markers present in the segment between two anchorswere averaged over different genetic linkage maps and calibr-ated according to the genetic distances between the two anchormarkers Third markers that were present on a few linkage mapswere incorporated relative to anchor markers Finally the mark-ers andgenes present onlyononeor twomapswere placedon theconsensus genetic maps via linked markers Genetic distanceson the consensus linkage maps are relative rather than absoluteIn cases where map distances between markers were not com-parable the distances were extrapolated based on an averageover majority of the maps

Nucleic Acids Research 2004 Vol 32 No 12 3547

Regions around the eukaryotic centromeres and in some casestelomeres have been reported to suppress recombinationrates (133031) Furthermore as shown in barley maize(Zea mays L) Arabidopsis and other plants distribution ofrecombination can be highly uneven even within a few kilobases of DNA (32ndash35) Relationships of the similar putativerecombination hotspots of wheat with the currently identifiedhighly recombinogenic chromosomal regions have not beenestablished

About 461 phenotypic markers and useful genes have beenidentified in wheat (36) (httpwheatpwusdagov) Althoughthe linkage relationship of about 377 has been establishedrelative to molecular markers the physical location of onlyseven of these genes is known (37) Because of uneven dist-ribution of recombination the physical location of the genesalong with a precise kbcM estimate for the region areessential for map-based cloning This is particularly importantfor wheat where the difference in the recombination amongregions is expected to be greater than other crop plants

The objectives of this study were to generate a comprehens-ive map of the wheat genome identify and precisely localizethe gene-containing regions reveal gene density distributionof recombination and kbcM estimate for each of thegene-containing regions and physically map phenotypicallycharacterized genes

MATERIALS AND METHODS

Plant material

Various aneuploid stocks along with the wild-type wheat culti-var Chinese Spring (CS) were used for physical mapping The21 nullisomicndashtetrasomic lines (NT missing a pair of chromo-somes the deficiency of which is compensated for by an extrapair of homoeologous chromosomes) were used for inter-chromosomal mapping and 14 ditelosomic lines (DT missinga pair of chromosome arms) were used to reveal arm location ofthe gene markers Sub-arm localization of markers was accom-plished using 334 deletion lines covering all 21 wheat chromo-somes A list of the deletion lines along with the fraction length(FL) of the retained arm is provided as supplementary informa-tion in Supplementary Table 1 These deletion lines weregenerated using gametocidal genes from Aegilops speltoides(38ndash40) Giemsa C-banding characterization and low-densityrestriction fragment length polymorphism (RFLP) mappingsuggested that most of these deletion lines resulted from singlebreaks followed by the loss of the acentric fragments (262740)High-density mapping revealed a few complex and secondarydeletionsrearrangements in some of the deletion lines (10)Additional information about the deletion lines is available athttpwwwksueduwgrcGermplasmDeletions

DNA analysis and marker selection

Plant genomic DNA was extracted following a previouslydescribed method (41) Gel blot analysis was performedusing 15 g of genomic DNA digested with either EcoRI orHindIII restriction enzymes and size separated on 08 agar-ose gels All other steps for the gel blot DNA analysis were aspreviously described (26)

The published and the Poaceae maps from the lsquograingenesrsquodatabase (httpwheatpwusdagovggpagesmapshtml) wereused to select gene markers for physical mapping The DNAmarkers were from wheat (FBA FBB NOR PSR TAGTAM UNL WG) Ae tauschii (KSU) barley (ABC ABGBCD MWG and cMWG) oat (CDO) and rice (RZ) (104243)Priority was given to cDNA clones although some PstI geno-mic clones were also used

Deletion mapping

Chromosomal and arm locations of each fragment band wererevealed by the NT and DT mapping A fragment band wasmapped to a chromosomal region bracketed by the breakpointsof the largest deletion possessing the fragment band and thesmallest deletion lacking it Multiple fragments were consid-ered non-allelic (shown by a letter at the end of the probename) if the corresponding bands mapped to different regionsor if more than two DNA fragments larger than the probe sizewere detected

Demarcating the gene-rich regions

The deletion mapping information for the three homoeologs ofeach group was combined to generate a high-resolution con-sensus physical map All deletion breakpoints for a group werealigned on a hypothetical chromosome based on FL values andeach marker was placed in the shortest possible interval basedon its location on the three homoeologs Due to the occasionaldifferences in size and distribution of gene-poor regionsamong homoeologs relative locations of markers were alsoconsidered along with the FL values to place deletion break-points on the consensus map To determine physical locationof a GRR the FL value consistent with at least two of thethree homoeologs was used Deletion mapping data fromthe three homoeologs along with FL location were used tolocalize deletion breakpoints within a GRR

Recombination analysis

A comprehensive genetic linkage map for each of the sevenwheat homoeologous groups was constructed using 137 geneticlinkage maps including 17 each for group 1 and 2 32 for 3 25 for4 16 for 5 and 15 each for group 6 and 7 chromosomes (httpwheatpwusdagovggpagesmapsshtml) First the markersgenes that were common among several wheat genetic linkagemaps for a homoeologous group were designated as anchorsTotal genetic length of the consensus map and distances amonganchors were estimated by taking average of recombinationamong anchor markers from various maps Second markerspresent between two anchor markers on multiple maps wereintegrated on the consensus map Relative genetic distancesof the markers present in the segment between two anchorswere averaged over different genetic linkage maps and calibr-ated according to the genetic distances between the two anchormarkers Third markers that were present on a few linkage mapswere incorporated relative to anchor markers Finally the mark-ers andgenes present onlyononeor twomapswere placedon theconsensus genetic maps via linked markers Genetic distanceson the consensus linkage maps are relative rather than absoluteIn cases where map distances between markers were not com-parable the distances were extrapolated based on an averageover majority of the maps

Nucleic Acids Research 2004 Vol 32 No 12 3547

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3548 Nucleic Acids Research 2004 Vol 32 No 12

Ta

ble

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Con

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Nucleic Acids Research 2004 Vol 32 No 12 3549

Ta

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68

4

Xcm

wg

716

Xcs

b1

12b

(Dh

n5

)X

fba1

X

fba4

2

Xfb

a8

1

Xfb

a1

11

X

fbb

40

X

fbb5

7

Xfb

b5

9

Xfb

b7

0

Xfb

b8

2

Xfb

b1

64

Xfb

b1

69

X

fbb1

70

X

fbb

22

1

Xfb

b3

27

X

ksu

D1

Xks

uD12

X

ksuD

17

Xks

uD27

X

ksuE

14

Xks

uF14

aX

ksuF

14b

Xks

uF19

X

ksuF

37

Xks

uG30

X

ksu

G51

X

ksuM

1X

ksuM

75

Xm

wg

19

Xm

wg

21

X

mw

g7

4a

Xm

wg

74

b

Xm

wg5

13

Xm

wg

514

X

mw

g7

98

X

mw

g813

X

mw

g838

X

mw

g887

X

mw

g897

X

mw

g934

X

mw

g2

053

X

mw

g2

10

0

Xp

sr2

(a-a

my1

)a

Xpsr

2(a

-am

y1)b

X

psr1

06b

Xps

r134

X

psr1

54

Xps

r546

X

psr6

05

Xpsr

90

4

Xpsr

90

8

Xps

r966

X

psr

96

7

Xrg

c74

Xta

g495

X

tag5

20

Xta

g547

X

tag6

80

Xta

g70

5

Xta

g762

X

tam

10

b

Xta

m21

X

tam

26

Xta

m2

8

Xta

m3

6

Xu

cd1

06

(Esi

18

)X

wg

34

1

Xw

g4

05

60

72

45

82

5

lsquo7S

09

rsquo7

AS

-1(0

90

)-1

00

7A

S-1

7

AS

-9P

gd3-

A

Xab

g378

X

abg7

04

Xb

cd9

3

Xbcd

13

0

Xb

cd5

88

Xb

cd9

07

Xbcd

18

72

X

cdo5

45

X

cdo

14

00

X

fba8

X

fba1

7X

fba6

5X

fba7

2X

fba9

3X

fba1

09

Xfb

b121

X

fba1

27

Xfb

a311

bX

fba3

21

Xfb

b9

Xfb

b156

X

fbb2

78

Xk

suD

9

Xm

wg5

30

Xm

wg

90

5

Xm

wg

20

80

X

psr

11

9

Xp

sr1

60(P

lc)

Xp

sr3

86

Xpsr

39

2

Xpsr

56

3

Xps

r648

X

psr8

33(P

er)

Xps

r946

aX

psr9

46b

Xta

g18

4

Xta

g6

1

Xum

c190

(Css

1)

Xu

nl2

22

X

un

l223

X

wye

835(

Wx)

30

32

40

14

81

lsquo7S

08

rsquo7

AS

-7(0

87

)-7

AS

-2(0

73

)7

AS

-7

7A

S-1

2

7A

S-2

Lr3

4-D

N

ar7-

A

Pc1

-B

Xab

c15

1

X

ab

c15

2

Xa

k4

66(N

ra)

Xbcd

14

38

X

cdo

43

1

X

cdo

47

5

Xcd

o5

34

Xcd

o6

76

Xcd

o7

80

X

cdo1

18

9

X

cdo

13

95

X

fba4

2X

fba3

46

Xfb

a377

X

fbb6

7a

Xfb

b186

aX

ksu

A1

Xk

suD

25

Xk

suE

3

Xksu

F4

8

Xk

suH

8

Xm

wg7

10

Xps

r490

(Ss1

-AB

D)

Xps

r604

X

psr6

62

Xrj

19

Xrz

2X

tam

13

Xta

g65

1

Xw

g83

4

23

22

36

19

78

lsquo7S

04

rsquo7

AS

-10

(04

5)-

7D

S-5

(03

6)

7D

S-1

7

DS

-5

7A

S-1

0

7A

S-5

Am

p-3-

A

Brz

-A

Est

3-A

P

an2-

A

Rc1

-A

Rc3

-D

Xa

bc4

65

Xbcd

31

0

Xbcd

38

5

Xb

cd7

07

Xb

cd9

81

Xb

cd1

06

6

Xbcd

13

38

X

bcd

17

73

X

cdo

57

X

cdo7

8

X

cdo9

1

X

cdo5

95

Xcm

wg

66

9

Xcm

wg

70

4

X

fba3

2X

fba2

48

Xfb

a340

X

fba3

71

Xfb

b150

X

fbb1

95

Xfb

b226

X

fbb2

64

Xk

suD

15

X

ksu

S1

X

mw

g8

9

Xpsr

10

3

Xp

sr1

08

Xp

sr1

50

Xp

sr1

52

Xps

r492

(Acl

3)

Xp

sr9

55

Xps

r192

1X

rj5

X

tam

64

X

un

l13

4

Xu

nl1

57

Xu

nl1

61

X

wg

18

0a

31

43

29

85

3

3552 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

C

onti

nued

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

ere

gio

nM

ark

ers

Gen

esbc

Gen

es(

)R

ec

()

Siz

e(M

b)

Rec

in

Kb

cM

lsquo7S

02

rsquo7

AS

-4(0

26

)-7

BS

-3(0

16

)7

AS

-6

7A

S-4

7

DS

-3

7B

S-3

Xa

bg

476

a

Xb

cd9

8

Xbcd

34

9

Xcm

wg

70

5

Xfb

a363

X

fbb3

43

Xm

wg

808

X

psr

65

X

rz4

X

tag

30

1

Xta

g53

6

Xta

g65

8

Xw

g5

22

Xw

g66

91

02

36

21

687

lsquo7L

01

rsquo7

DL

-6(0

10

)-7

AL

-4(0

18

)7

DL

-6

7A

L-4

7

BL

-14

Xcd

o5

51

X

cdo1

19

9

Xpsr

16

9

Xp

sr3

03

Xta

g43

9

Xta

g64

23

13

31

52

78

lsquo7L

03

rsquo7

AL

-3(0

29

)-7

BL

-1(0

40

)7

AL

-3

7B

L-2

7

BL

-1

7D

L-5

Am

y2-A

V

Atp

-B1-

A

Xab

c455

X

ab

g4

76

b

Xcd

o5

3

Xcd

o6

73

X

cdo

92

0

Xcd

o1

40

7

Xcd

o1

42

8

Xh

r20b

X

mw

g2

03

1

Xm

wg8

25

Xpsr

10

5

Xpsr

16

5

Xp

sr3

11

Xp

sr3

40

Xp

sr3

89

Xps

r468

(Pep

c)

Xpsr

51

4

Xpsr

69

0

Xps

r803

(Fed

)X

psr

93

8

Xta

g5

98

X

ubp2

X

un

l188

X

wg

46

6

Xw

g68

6a

X

wg

71

9

Xw

ye19

58(A

dpg3

)

19

54

51

38

89

lsquo7L

08

rsquo7

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-4(0

76

)-7

AL

-2(0

87

)7

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-21

7

AL

-6

7B

L-1

0

7B

L-1

3

7A

L-8

7

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-13

7A

L-1

6

7A

L-2

7

DL

-4

Ep-

D1b

-D

Lr1

9-D

P

m5-

B

Sr22

-A

VA

tp-A

-A

Xab

c305

X

ab

c31

0

Xbc

d178

X

bcd

19

30

X

cdo6

65

X

cdo6

86

Xcd

o7

75

Xcd

o962

X

fba6

9X

fba9

7X

fba2

04

Xfb

a234

X

fba2

59

Xfb

a264

X

fba3

01

Xfb

a350

X

fba3

82

Xfb

b67b

X

fbb7

9X

fbb1

75

Xfb

b218

X

fbb3

25

Xks

u927

(Grp

94a)

X

ksu9

27(G

rp94

b)

Xksu

A5

X

ksu

D2

X

ksu

D39

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ksu

D6

Xk

suD

7

Xk

suD

9

Xksu

G1

2

Xk

suG

39

Xk

suG

7

Xm

wg

97

5

Xpsr

72

X

psr

11

7

Xp

sr1

29

Xps

r350

aX

psr3

50b

Xpsr

54

7

Xp

sr5

48

Xpsr

56

0

Xpsr

68

1

Xpsr

92

7

Xri

s44

Xrr

11

X

rz4

76

X

rz6

82

X

tag

47

8

Xta

g5

49

X

tag

68

6

Xta

m6

2a

Xta

m6

2b

Xuc

d101

(Esi

2)

Xu

nl1

70

X

un

l185

X

un

l204

X

wg

18

0b

X

wg4

20

Xw

g514

X

wg

68

6b

43

39

41

86

3

lsquo7L

10

rsquo7

AL

-9(0

94

)-7

AL

-15(0

99

)7

AL

-9

7A

L-1

5B

dv1

Cht

1b-B

D

Ep-

V1

Lr1

4a-B

L

r20-

A

Pch

1-A

BD

P

ch2-

A

Pm

1-A

P

m9-

A

Sr15

-A

Sr17

-B

Xab

g461

X

cdo3

47

X

cdo

41

4

Xcs

ih8

1

Xfb

b18

Xfb

a21

Xfb

a134

X

fbb1

45

Xfb

b186

bX

fbb1

89

Xfb

b238

X

ksu

929(

Cat

)X

ksu

B7

Xks

uE18

X

ksu

G3

4

Xks

uH9

Xm

wg9

38

Xm

wg

206

2

Xp

sr1

21(G

lb3)

Xpsr

14

8

Xps

r680

X

psr

68

7

Xp

sr7

43

Xps

r965

X

psr1

923

Xps

r195

4(T

ha)

Xrz

508

Xta

g3

5

Xta

g54

6

Xta

g75

0

Xta

m50

X

wg

34

1

Xw

g3

80

X

wg

86

6

Xw

g18

06

X

wia

482

31

53

21

44

2

aM

ajor

GR

Rs

are

un

der

lin

ed

bA

ster

isk

repre

sen

tm

ark

ers

ten

tati

vel

yp

lace

dw

ith

ina

GR

R

cM

ark

ers

inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

mar

ker

sar

eb

oth

pre

sen

to

nth

eco

nse

nsu

sp

hy

sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

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ble

1

Con

tinu

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GR

Rsa

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Del

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)R

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89

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r16-

D

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703

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d18

Xbc

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d718

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bcd1

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Xcd

o57

Xcd

o447

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cdo4

56a

Xcd

o456

bX

cdo4

56c

Xcd

o109

0X

cmw

g682

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fba4

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fba2

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fba7

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Xfb

a70b

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fba8

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fba8

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fba3

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Xfb

b40

Xfb

b61

Xfb

b62a

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fbb1

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Xfb

b289

X

ksu

D1

8

Xpsr

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9

Xps

r566

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psr6

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Xp

sr6

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Xps

r908

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psr9

33

Xps

r958

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tag1

97

Xta

g302

X

un

l14

5

X

un

l216

X

un

lBE

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28

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Xu

nlB

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97

251

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un

lBE

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95

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lBE

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70

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lBF

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Xu

nlB

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685

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lsquo2S

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BS

-14(0

84

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BS

-3(0

79

)2

BS

-14

2B

S-3

Lr2

3-B

L

r-2A

L

r15-

A

Lr2

8-A

Sr

6-A

X

ab

g2

X

bcd2

62

Xb

cd3

48

Xbc

d611

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bcd

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84

X

bcd1

688

Xbc

d170

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bs12

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Xcd

o4

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cdo7

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o128

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379

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o147

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Xfb

a178

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fba2

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Xfb

a300

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fbb2

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b329

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fbb3

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Xks

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(Per

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bp)

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19-A

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21-A

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36-A

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bcd1

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Xbc

d120

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Xbc

d161

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d111

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fbb0

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Xfb

b021

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b353

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Xpsr

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395

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DL

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26

)-2

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-2(0

36

)2

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-4

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L-3

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BL

-2X

bcd4

45

Xcd

o3

70

Xfb

a199

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fbb3

35

Xp

sr1

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Xps

r135

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psr

38

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Xps

r489

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l11

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-3

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a74

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Xps

r129

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r575

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be)

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lsquo2L

08

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AL

-3(0

77

)-2

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-1(0

85

)2

AL

-3

2A

L-1

Xbcd

13

5

X

bcd3

07

Xfb

a62

Xfb

a64

Xfb

a111

X

fba3

41b

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22

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29

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bp2)

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bp1)

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ksu9

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hi1)

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ksu9

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ksu

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r331

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psr3

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r540

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r644

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r692

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r932

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r956

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tag2

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2-B

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rns

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fr-D

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lsquo7L

08

rsquo7

DL

-4(0

76

)-7

AL

-2(0

87

)7

AL

-21

7

AL

-6

7B

L-1

0

7B

L-1

3

7A

L-8

7

AL

-13

7A

L-1

6

7A

L-2

7

DL

-4

Ep-

D1b

-D

Lr1

9-D

P

m5-

B

Sr22

-A

VA

tp-A

-A

Xab

c305

X

ab

c31

0

Xbc

d178

X

bcd

19

30

X

cdo6

65

X

cdo6

86

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o7

75

Xcd

o962

X

fba6

9X

fba9

7X

fba2

04

Xfb

a234

X

fba2

59

Xfb

a264

X

fba3

01

Xfb

a350

X

fba3

82

Xfb

b67b

X

fbb7

9X

fbb1

75

Xfb

b218

X

fbb3

25

Xks

u927

(Grp

94a)

X

ksu9

27(G

rp94

b)

Xksu

A5

X

ksu

D2

X

ksu

D39

X

ksu

D6

Xk

suD

7

Xk

suD

9

Xksu

G1

2

Xk

suG

39

Xk

suG

7

Xm

wg

97

5

Xpsr

72

X

psr

11

7

Xp

sr1

29

Xps

r350

aX

psr3

50b

Xpsr

54

7

Xp

sr5

48

Xpsr

56

0

Xpsr

68

1

Xpsr

92

7

Xri

s44

Xrr

11

X

rz4

76

X

rz6

82

X

tag

47

8

Xta

g5

49

X

tag

68

6

Xta

m6

2a

Xta

m6

2b

Xuc

d101

(Esi

2)

Xu

nl1

70

X

un

l185

X

un

l204

X

wg

18

0b

X

wg4

20

Xw

g514

X

wg

68

6b

43

39

41

86

3

lsquo7L

10

rsquo7

AL

-9(0

94

)-7

AL

-15(0

99

)7

AL

-9

7A

L-1

5B

dv1

Cht

1b-B

D

Ep-

V1

Lr1

4a-B

L

r20-

A

Pch

1-A

BD

P

ch2-

A

Pm

1-A

P

m9-

A

Sr15

-A

Sr17

-B

Xab

g461

X

cdo3

47

X

cdo

41

4

Xcs

ih8

1

Xfb

b18

Xfb

a21

Xfb

a134

X

fbb1

45

Xfb

b186

bX

fbb1

89

Xfb

b238

X

ksu

929(

Cat

)X

ksu

B7

Xks

uE18

X

ksu

G3

4

Xks

uH9

Xm

wg9

38

Xm

wg

206

2

Xp

sr1

21(G

lb3)

Xpsr

14

8

Xps

r680

X

psr

68

7

Xp

sr7

43

Xps

r965

X

psr1

923

Xps

r195

4(T

ha)

Xrz

508

Xta

g3

5

Xta

g54

6

Xta

g75

0

Xta

m50

X

wg

34

1

Xw

g3

80

X

wg

86

6

Xw

g18

06

X

wia

482

31

53

21

44

2

aM

ajor

GR

Rs

are

un

der

lin

ed

bA

ster

isk

repre

sen

tm

ark

ers

ten

tati

vel

yp

lace

dw

ith

ina

GR

R

cM

ark

ers

inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

mar

ker

sar

eb

oth

pre

sen

to

nth

eco

nse

nsu

sp

hy

sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

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3552 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

C

onti

nued

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

ere

gio

nM

ark

ers

Gen

esbc

Gen

es(

)R

ec

()

Siz

e(M

b)

Rec

in

Kb

cM

lsquo7S

02

rsquo7

AS

-4(0

26

)-7

BS

-3(0

16

)7

AS

-6

7A

S-4

7

DS

-3

7B

S-3

Xa

bg

476

a

Xb

cd9

8

Xbcd

34

9

Xcm

wg

70

5

Xfb

a363

X

fbb3

43

Xm

wg

808

X

psr

65

X

rz4

X

tag

30

1

Xta

g53

6

Xta

g65

8

Xw

g5

22

Xw

g66

91

02

36

21

687

lsquo7L

01

rsquo7

DL

-6(0

10

)-7

AL

-4(0

18

)7

DL

-6

7A

L-4

7

BL

-14

Xcd

o5

51

X

cdo1

19

9

Xpsr

16

9

Xp

sr3

03

Xta

g43

9

Xta

g64

23

13

31

52

78

lsquo7L

03

rsquo7

AL

-3(0

29

)-7

BL

-1(0

40

)7

AL

-3

7B

L-2

7

BL

-1

7D

L-5

Am

y2-A

V

Atp

-B1-

A

Xab

c455

X

ab

g4

76

b

Xcd

o5

3

Xcd

o6

73

X

cdo

92

0

Xcd

o1

40

7

Xcd

o1

42

8

Xh

r20b

X

mw

g2

03

1

Xm

wg8

25

Xpsr

10

5

Xpsr

16

5

Xp

sr3

11

Xp

sr3

40

Xp

sr3

89

Xps

r468

(Pep

c)

Xpsr

51

4

Xpsr

69

0

Xps

r803

(Fed

)X

psr

93

8

Xta

g5

98

X

ubp2

X

un

l188

X

wg

46

6

Xw

g68

6a

X

wg

71

9

Xw

ye19

58(A

dpg3

)

19

54

51

38

89

lsquo7L

08

rsquo7

DL

-4(0

76

)-7

AL

-2(0

87

)7

AL

-21

7

AL

-6

7B

L-1

0

7B

L-1

3

7A

L-8

7

AL

-13

7A

L-1

6

7A

L-2

7

DL

-4

Ep-

D1b

-D

Lr1

9-D

P

m5-

B

Sr22

-A

VA

tp-A

-A

Xab

c305

X

ab

c31

0

Xbc

d178

X

bcd

19

30

X

cdo6

65

X

cdo6

86

Xcd

o7

75

Xcd

o962

X

fba6

9X

fba9

7X

fba2

04

Xfb

a234

X

fba2

59

Xfb

a264

X

fba3

01

Xfb

a350

X

fba3

82

Xfb

b67b

X

fbb7

9X

fbb1

75

Xfb

b218

X

fbb3

25

Xks

u927

(Grp

94a)

X

ksu9

27(G

rp94

b)

Xksu

A5

X

ksu

D2

X

ksu

D39

X

ksu

D6

Xk

suD

7

Xk

suD

9

Xksu

G1

2

Xk

suG

39

Xk

suG

7

Xm

wg

97

5

Xpsr

72

X

psr

11

7

Xp

sr1

29

Xps

r350

aX

psr3

50b

Xpsr

54

7

Xp

sr5

48

Xpsr

56

0

Xpsr

68

1

Xpsr

92

7

Xri

s44

Xrr

11

X

rz4

76

X

rz6

82

X

tag

47

8

Xta

g5

49

X

tag

68

6

Xta

m6

2a

Xta

m6

2b

Xuc

d101

(Esi

2)

Xu

nl1

70

X

un

l185

X

un

l204

X

wg

18

0b

X

wg4

20

Xw

g514

X

wg

68

6b

43

39

41

86

3

lsquo7L

10

rsquo7

AL

-9(0

94

)-7

AL

-15(0

99

)7

AL

-9

7A

L-1

5B

dv1

Cht

1b-B

D

Ep-

V1

Lr1

4a-B

L

r20-

A

Pch

1-A

BD

P

ch2-

A

Pm

1-A

P

m9-

A

Sr15

-A

Sr17

-B

Xab

g461

X

cdo3

47

X

cdo

41

4

Xcs

ih8

1

Xfb

b18

Xfb

a21

Xfb

a134

X

fbb1

45

Xfb

b186

bX

fbb1

89

Xfb

b238

X

ksu

929(

Cat

)X

ksu

B7

Xks

uE18

X

ksu

G3

4

Xks

uH9

Xm

wg9

38

Xm

wg

206

2

Xp

sr1

21(G

lb3)

Xpsr

14

8

Xps

r680

X

psr

68

7

Xp

sr7

43

Xps

r965

X

psr1

923

Xps

r195

4(T

ha)

Xrz

508

Xta

g3

5

Xta

g54

6

Xta

g75

0

Xta

m50

X

wg

34

1

Xw

g3

80

X

wg

86

6

Xw

g18

06

X

wia

482

31

53

21

44

2

aM

ajor

GR

Rs

are

un

der

lin

ed

bA

ster

isk

repre

sen

tm

ark

ers

ten

tati

vel

yp

lace

dw

ith

ina

GR

R

cM

ark

ers

inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

mar

ker

sar

eb

oth

pre

sen

to

nth

eco

nse

nsu

sp

hy

sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

C

onti

nued

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

ere

gio

nM

arker

sG

enes

bc

Gen

es(

)R

ec

()

Siz

e(M

b)

Rec

in

Kb

cM

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09

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-6(0

97

)-5

BS

-6(0

81

)5

AS

-6

5B

S-6

Gsp

-AB

D

Ha-

D

Xa

bg

479

X

ab

g7

05

Xbcd

87

3

Xcd

o959

X

fba1

14

X

fba

36

7

Xfb

b1

21

X

fbb

27

6

Xfb

b277

X

mw

g8

35

X

psr

17

0

Xps

r618

X

psr2

152b

X

tag

31

7

Xw

g687

29

60

43

23

89

lsquo5S

07

rsquo5

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-3(0

75

)-5

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-5(0

67

)5

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S-5

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r-A

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m2-

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Xb

cd1

871

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68

7

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o7

49

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fba1

31

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a2

32

X

fba

34

2

Xfb

a352

X

fba3

93

X

ksu9

22(T

ha)

X

psr

32

6

Xpsr

62

8

Xpsr

94

0a

X

psr

94

0b

X

psr

94

5

Xta

g31

9

Xta

g62

9

Xta

m5

3a

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m5

3b

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bp

3

Xu

bp

5

Xuc

w14

1

39

28

21

30

00

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06

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56

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43

)5

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-8

5D

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11

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X

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44

X

cdo

45

9a

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cdo

45

9b

X

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77

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o6

77

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o1

33

5

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suI2

6

Xpsr

11

8

Xp

sr9

29

Xp

sr1

204

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rg4

63

Xrg

90

1

Xta

m5

42

45

19

63

33

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rsquo

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-2

5B

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X

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H8

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11

8

Xpsr

90

3

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r946

X

wg

34

1

Xw

g3

63

14

31

34

33

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15

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20

4

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087

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o785

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9

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327

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68

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bg

314

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g3

87

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ab

g4

73

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9

X

bcd

21

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bcd

15

7

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bcd

30

7a

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bcd

35

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bcd

35

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X

bcd

50

8

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Xb

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088

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140

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gb1

0

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gb

63

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b1

74

X

mg

b1

91

X

mg

b3

01

X

mg

b3

41

X

mw

g5

2

Xm

wg

55

2

Xm

wg

58

3

Xm

wg

592

X

mw

g6

04

Xm

wg6

24

Xm

wg

76

8

Xm

wg

914

Xm

wg

923

X

mw

g9

56

a

Xm

wg

95

6b

X

psr

10

0

Xpsr

12

0b

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r492

(Acl

3)

Xps

r637

X

psr8

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X

psr9

11

Xps

r912

X

psr1

202

Xta

g61

4

Xta

g6

44

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bp

25

X

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04(E

si14

)X

un

l4

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nl1

04

X

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l109

X

un

l113

X

un

l116

X

un

l117

X

un

l119

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l12

1

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nl1

23

X

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l13

8

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nl1

39

X

un

l14

1

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nl1

52

Xu

nl1

60

Xu

nl1

69

X

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l191

X

un

l21

3

Xu

nl2

14

X

un

l21

7

Xu

nl2

18

X

un

l22

4

Xw

g3

64

X

wg

53

0

Xw

g56

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Xw

g8

89

Xw

g102

6

36

20

61

20

33

lsquo5L

08

rsquo5

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-14

(07

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79

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Nucleic Acids Research 2004 Vol 32 No 12 3551

Ta

ble

1

Con

tinu

ed

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

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Gen

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mw

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3552 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

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onti

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GR

Rsa

Fla

nk

ing

del

etio

ns

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etio

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inth

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wg

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wg

86

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aM

ajor

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repre

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ten

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lace

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ith

ina

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R

cM

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inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

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pre

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to

nth

eco

nse

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sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

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r149

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091

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64

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b1

69

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fbb1

70

X

fbb

22

1

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b3

27

X

ksu

D1

Xks

uD12

X

ksuD

17

Xks

uD27

X

ksuE

14

Xks

uF14

aX

ksuF

14b

Xks

uF19

X

ksuF

37

Xks

uG30

X

ksu

G51

X

ksuM

1X

ksuM

75

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wg

19

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wg

21

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mw

g7

4a

Xm

wg

74

b

Xm

wg5

13

Xm

wg

514

X

mw

g7

98

X

mw

g813

X

mw

g838

X

mw

g887

X

mw

g897

X

mw

g934

X

mw

g2

053

X

mw

g2

10

0

Xp

sr2

(a-a

my1

)a

Xpsr

2(a

-am

y1)b

X

psr1

06b

Xps

r134

X

psr1

54

Xps

r546

X

psr6

05

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90

4

Xpsr

90

8

Xps

r966

X

psr

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7

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c74

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g495

X

tag5

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Xta

g547

X

tag6

80

Xta

g70

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g762

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tam

10

b

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m21

X

tam

26

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m2

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Xta

m3

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(Esi

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)X

wg

34

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g4

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60

72

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82

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09

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g378

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07

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72

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cdo5

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cdo

14

00

X

fba8

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fba1

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fba6

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fba7

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fba9

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09

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b121

X

fba1

27

Xfb

a311

bX

fba3

21

Xfb

b9

Xfb

b156

X

fbb2

78

Xk

suD

9

Xm

wg5

30

Xm

wg

90

5

Xm

wg

20

80

X

psr

11

9

Xp

sr1

60(P

lc)

Xp

sr3

86

Xpsr

39

2

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56

3

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r648

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psr8

33(P

er)

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r946

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g18

4

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g6

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c190

(Css

1)

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nl2

22

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l223

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wye

835(

Wx)

30

32

40

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81

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08

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87

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73

)7

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-7

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c15

1

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c15

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66(N

ra)

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14

38

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cdo

43

1

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cdo

47

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o5

34

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o6

76

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o7

80

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cdo1

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9

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cdo

13

95

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fba4

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46

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a377

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fbb6

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25

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8

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suH

8

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wg7

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r490

(Ss1

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r604

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62

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tam

13

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g65

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g83

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04

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6)

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7

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-5

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7A

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p-3-

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-A

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bc4

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81

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cd1

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6

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13

38

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bcd

17

73

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cdo

57

X

cdo7

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X

cdo9

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cdo5

95

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wg

66

9

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wg

70

4

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fba3

2X

fba2

48

Xfb

a340

X

fba3

71

Xfb

b150

X

fbb1

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b226

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fbb2

64

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suD

15

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ksu

S1

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mw

g8

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10

3

Xp

sr1

08

Xp

sr1

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sr1

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r492

(Acl

3)

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sr9

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r192

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tam

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nl1

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wg

18

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85

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3552 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

C

onti

nued

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

ere

gio

nM

ark

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Gen

esbc

Gen

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)R

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()

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Rec

in

Kb

cM

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02

rsquo7

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26

)-7

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16

)7

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-3

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S-3

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bg

476

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cd9

8

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34

9

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wg

70

5

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a363

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fbb3

43

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wg

808

X

psr

65

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rz4

X

tag

30

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g53

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g65

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g5

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g66

91

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01

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10

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18

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r20b

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c31

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o962

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fba2

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fba3

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97

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92

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tag

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wg

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10

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94

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g461

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cdo

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r680

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sr7

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g75

0

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wg

34

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g3

80

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wg

86

6

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g18

06

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wia

482

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aM

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repre

sen

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ark

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ten

tati

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lace

dw

ith

ina

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R

cM

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ers

inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

mar

ker

sar

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oth

pre

sen

to

nth

eco

nse

nsu

sp

hy

sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

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92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

Tab

le1

C

onti

nued

GR

Rsa

Fla

nk

ing

del

etio

ns

Del

etio

ns

inth

ere

gio

nM

ark

ers

Gen

esbc

Gen

es(

)R

ec

()

Siz

e(M

b)

Rec

in

Kb

cM

lsquo7S

02

rsquo7

AS

-4(0

26

)-7

BS

-3(0

16

)7

AS

-6

7A

S-4

7

DS

-3

7B

S-3

Xa

bg

476

a

Xb

cd9

8

Xbcd

34

9

Xcm

wg

70

5

Xfb

a363

X

fbb3

43

Xm

wg

808

X

psr

65

X

rz4

X

tag

30

1

Xta

g53

6

Xta

g65

8

Xw

g5

22

Xw

g66

91

02

36

21

687

lsquo7L

01

rsquo7

DL

-6(0

10

)-7

AL

-4(0

18

)7

DL

-6

7A

L-4

7

BL

-14

Xcd

o5

51

X

cdo1

19

9

Xpsr

16

9

Xp

sr3

03

Xta

g43

9

Xta

g64

23

13

31

52

78

lsquo7L

03

rsquo7

AL

-3(0

29

)-7

BL

-1(0

40

)7

AL

-3

7B

L-2

7

BL

-1

7D

L-5

Am

y2-A

V

Atp

-B1-

A

Xab

c455

X

ab

g4

76

b

Xcd

o5

3

Xcd

o6

73

X

cdo

92

0

Xcd

o1

40

7

Xcd

o1

42

8

Xh

r20b

X

mw

g2

03

1

Xm

wg8

25

Xpsr

10

5

Xpsr

16

5

Xp

sr3

11

Xp

sr3

40

Xp

sr3

89

Xps

r468

(Pep

c)

Xpsr

51

4

Xpsr

69

0

Xps

r803

(Fed

)X

psr

93

8

Xta

g5

98

X

ubp2

X

un

l188

X

wg

46

6

Xw

g68

6a

X

wg

71

9

Xw

ye19

58(A

dpg3

)

19

54

51

38

89

lsquo7L

08

rsquo7

DL

-4(0

76

)-7

AL

-2(0

87

)7

AL

-21

7

AL

-6

7B

L-1

0

7B

L-1

3

7A

L-8

7

AL

-13

7A

L-1

6

7A

L-2

7

DL

-4

Ep-

D1b

-D

Lr1

9-D

P

m5-

B

Sr22

-A

VA

tp-A

-A

Xab

c305

X

ab

c31

0

Xbc

d178

X

bcd

19

30

X

cdo6

65

X

cdo6

86

Xcd

o7

75

Xcd

o962

X

fba6

9X

fba9

7X

fba2

04

Xfb

a234

X

fba2

59

Xfb

a264

X

fba3

01

Xfb

a350

X

fba3

82

Xfb

b67b

X

fbb7

9X

fbb1

75

Xfb

b218

X

fbb3

25

Xks

u927

(Grp

94a)

X

ksu9

27(G

rp94

b)

Xksu

A5

X

ksu

D2

X

ksu

D39

X

ksu

D6

Xk

suD

7

Xk

suD

9

Xksu

G1

2

Xk

suG

39

Xk

suG

7

Xm

wg

97

5

Xpsr

72

X

psr

11

7

Xp

sr1

29

Xps

r350

aX

psr3

50b

Xpsr

54

7

Xp

sr5

48

Xpsr

56

0

Xpsr

68

1

Xpsr

92

7

Xri

s44

Xrr

11

X

rz4

76

X

rz6

82

X

tag

47

8

Xta

g5

49

X

tag

68

6

Xta

m6

2a

Xta

m6

2b

Xuc

d101

(Esi

2)

Xu

nl1

70

X

un

l185

X

un

l204

X

wg

18

0b

X

wg4

20

Xw

g514

X

wg

68

6b

43

39

41

86

3

lsquo7L

10

rsquo7

AL

-9(0

94

)-7

AL

-15(0

99

)7

AL

-9

7A

L-1

5B

dv1

Cht

1b-B

D

Ep-

V1

Lr1

4a-B

L

r20-

A

Pch

1-A

BD

P

ch2-

A

Pm

1-A

P

m9-

A

Sr15

-A

Sr17

-B

Xab

g461

X

cdo3

47

X

cdo

41

4

Xcs

ih8

1

Xfb

b18

Xfb

a21

Xfb

a134

X

fbb1

45

Xfb

b186

bX

fbb1

89

Xfb

b238

X

ksu

929(

Cat

)X

ksu

B7

Xks

uE18

X

ksu

G3

4

Xks

uH9

Xm

wg9

38

Xm

wg

206

2

Xp

sr1

21(G

lb3)

Xpsr

14

8

Xps

r680

X

psr

68

7

Xp

sr7

43

Xps

r965

X

psr1

923

Xps

r195

4(T

ha)

Xrz

508

Xta

g3

5

Xta

g54

6

Xta

g75

0

Xta

m50

X

wg

34

1

Xw

g3

80

X

wg

86

6

Xw

g18

06

X

wia

482

31

53

21

44

2

aM

ajor

GR

Rs

are

un

der

lin

ed

bA

ster

isk

repre

sen

tm

ark

ers

ten

tati

vel

yp

lace

dw

ith

ina

GR

R

cM

ark

ers

inb

old

are

ph

ysi

call

ym

app

ed

Th

eu

nd

erli

ned

mar

ker

sar

eb

oth

pre

sen

to

nth

eco

nse

nsu

sp

hy

sica

lan

dg

enet

icm

aps

Nucleic Acids Research 2004 Vol 32 No 12 3553

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

RESULTS

In order to identify and demarcate the gene-containing regionsprecise physical location of 3025 gene marker loci including252 phenotypically characterized genes and 17 quantitativetrait loci (QTLs) was revealed First 942 cDNA or PstI geno-mic clones were physically mapped using 334 deletion linesEach gene marker loci was localized to the smallest possiblechromosomal interval by combining mapping informationfrom the three wheat homoeologs on a consensus physicalmap This analysis localized the gene-containing regionsand identified the flanking deletion breakpoints Second aconsensus genetic linkage map was constructed by combininginformation from 137 wheat maps and was compared with theconsensus physical map via 428 common markers (underlinedbold face markers Table 1) This comparison revealed thephysical location of phenotypically characterized genes con-firmed gene distribution and provided estimates of recombina-tion at a sub-chromosomal level

Physical mapping

The physical mapping results are provided in supplementaryfigures (AndashG) and are summarized in Table 1 With an averageof 135 the number of physically mapped probes ranged from43 for group 4 to 274 for group 5 chromosomes The averagenumber of deletion lines used per homoeologous group was 48with a range of 40 to 58 (Table 2) The 942 probes detected2036 loci of which 617 were for the A 740 for the B and 679were for the D genome chromosomes (Table 3) With anaverage of 22 the number of loci per probe ranged from19 for group 6 to 24 for groups 1 and 3 Using one restrictionenzyme 45 of the probes detected loci on all three homo-eologs 25 on two and 29 of the probes detected loci ononly one of the three homoeologs (Table 3) Among the probesdetecting loci on two homoeologs the number for B and Dgenome chromosomes was more than double (124 probes)compared to either of the other two combinations (A and Dor A and B) Similarly among the probes detecting loci on onlyone of the three homoeologs the number for the B genome wasmore than double compared to either of the other two Thetotal number of loci detected for the B genome was 37compared with 33 for the D and 30 for the A genomeThe number of probes mapping on all three homoeologs ran-ged from 29 for group 6 to 60 for group 1 Similarly thenumber of probes mapping only on one of the three homoeo-logs ranged from 19 for group 3 to 41 for group 6

Except for a few discrepancies relative order FL locationand overall distribution of gene markers were similar amonghomoeologs It was therefore possible to generate consensusphysical maps by combining mapping information from thehomoeologs Deletion breakpoints of homoeologs were placedon the consensus map and each marker was placed to theshortest possible interval (see Methods)

Gene distribution on the wheat chromosomes is obviousfrom Figure 1 Regions of very high marker density wereobserved on all consensus physical maps and were calledgene-rich regions (GRRs) The location and size of eachGRR was drawn to scale and the bracketing deletion break-points are shown on the lsquoleftrsquo Detailed information regardingthe bracketing deletions size markers and other deletion linesfor each GRR is given in Table 1 and is summarized in Table 2With an average of seven and a range of five to eight perchromosome 48 GRRs were identified for the wheat genomethat contained 94 of the markers (Table 2) The remaining6 of the markers were present in other regions includingcentromere of chromosome 1B that contained markersXbcd1072 and Xpsr161

Comparative analysis

With the objectives to confirm the gene distribution physicallymap additional markers including phenotypically character-ized genes and to reveal precise distribution of recombinationon the wheat chromosomes consensus genetic linkage mapswere constructed for each of the homoeologous groups andcompared with the consensus physical maps A total of 1417markers including 252 phenotypically characterized genes and17 QTLs were placed on the consensus genetic linkage maps(Figure 1) With an average of 202 per chromosome thenumber of markers ranged from 151 for group 4 to 235 forgroup 2 The average number of phenotypically characterizedgenesQTLs was 38 per chromosome group with a range of 28for group 5 to 43 for group 1

The 428 common markers were used to compare the con-sensus physical and genetic linkage maps A sector corre-sponding to each of the GRR was identified on the geneticlinkage map Additional markers and useful genes present onthe consensus linkage map were then localized to the corre-sponding GRR and the results are summarized in Table 1About 94 (13361417) of the consensus genetic linkagemap markers including 241 of the 252 phenotypically char-acterized genes and all 17 QTLs mapped in the GRRs(Figure 1 Table 1) The distribution of gene markers observed

Table 2 Summary information about the identified GRRs

Group Size (Mb) No of del in GRR Total No of deletion No of Marker No of GRRS L Total S L Total S L Total S L Total S L Total

1 25 94 119 19 32 51 26 26 52 126 126 252 3 5 82 67 111 178 8 8 16 25 17 42 128 145 273 3 4 73 131 131 262 9 10 19 20 20 40 88 153 241 3 4 74 94 167 261 5 18 23 16 33 49 56 106 162 2 3 55 96 192 288 11 18 29 17 35 52 62 290 352 4 4 86 51 84 135 14 22 36 15 26 41 85 163 248 2 3 57 141 140 281 13 18 31 19 39 58 137 154 291 4 4 8Total 605 919 1524 79 126 205 138 196 334 682 1137 1819 21 27 48

3554 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

from the genetic linkage maps was similar to that from thephysical maps Between the two methods 3025 marker lociwere physically localized on wheat chromosomes that corre-sponded to 1931 unique gene loci (Figure 1 Table 1) Of these94 mapped within the 48 GRRs The remaining 6 werepresent in small groups of one to four markers separated bymore than one deletion breakpoints

Gene distribution

The number and location of GRRs was different among wheatchromosomes (Figure 1 and Table 1) Twenty-one GRRs werepresent on the short arms and contained 35 of the wheatgenes The remaining 27 GRRs were present on the long armsand contained about 59 of the genes (Table 1) The numberof GRRs per chromosome varied from five (groups 4 and 6) toeight (Groups 1 5 and 7) Essentially no GRR was observed inthe centromeric regions of the chromosomes Only one smallGRR (lsquo7L01rsquo) containing 4 of the armrsquos genes was observedin the proximal 20 of any chromosome In general GRRspresent in the distal regions were relatively higher in genedensity More than 80 of the total marker loci mapped inthe distal half of the chromosomes and 58 mapped in thedistal 20 (Figure 1 and Table 1)

Significant differences were observed among GRRs formarker number marker density and the size The 48 GRRsencompassed about 1554 Mb that is equivalent to 29 of thewheat genome (Table 1) Gene density and number in18 GRRs was very high (major GRRs) (underlined inTable 1 and Figure 1) These major GRRs contained nearly60 of the wheat genes but covered only 11 of the genomeThe gene number and density was variable even among thesemajor GRRs Five of these (lsquo1S08rsquo lsquo2L10rsquo lsquo4S07rsquo lsquo6S10rsquoand lsquo6L09rsquo) contained 26 of the wheat genes but spannedonly 3 of the genome On the contrary four minor GRRs(lsquo1S04rsquo lsquo2L03rsquo lsquo5S04rsquo and lsquo7L01rsquo) contained only 2 ofthe wheat genes but covered gt2 of the genome Estimatedsizes of GRRs varied from 3 Mb for lsquo1S04rsquo to 71 Mb forlsquo3L09rsquo with an average of 32 Mb The total region spannedby GRRs ranged from 119 Mb for group 1 to 288 Mb forgroup 5 Number of genes in a GRR varied from 4 of the armin lsquo7L01rsquo to 82 in lsquo6S10rsquo Gene density varied as much as70-fold among GRRs with a range from a gene per 78 kb inlsquo1S08rsquo to 5500 kb in lsquo7L01rsquo Gene distribution even withinGRRs appeared to be uneven The lsquo1L09rsquo region is presentbetween FLs 084 and 09 Of the total 22 markers present inthe region 16 mapped between FL 084 and 085 one betweenFL 085 and 089 and five between FL 089 and 09

Physical mapping of phenotypic markers

The 269 phenotypic markers mapped in this study included80 monogenically inherited wheat disease resistance genes and17 QTLs (Figure 1 and Table 1) Some of the other usefulgenes were Eps (earliness per se) Hd (awnedness) Kr (cross-ibility) Ph1 plant height male sterility and ear morphologygenes (36) Of the 269 phenotypic markers and useful genes159 were reliably located on the consensus genetic maps(colored red Figure 1) The remaining 93 genes were placedon the consensus genetic maps because of their close associa-tion (lt5 cM) with one or more markers The 17 QTLs werelocated to 10 cM intervals on the consensus linkage map asT

ab

le3

S

um

mar

yo

fth

ep

hy

sica

lly

map

ped

wh

eat

loci

on

ho

mo

eolo

go

us

gro

ups

Chro

mo

som

e

gro

up

s

AB

DA

BB

DA

DA

BD

LN

o

PL

PL

PL

PL

PL

PL

PL

To

tal

AT

ota

lB

To

tal

CT

ota

lP

ML

To

tal

PM

LC

AG

ran

dto

tal

18

92

67

36

19

38

12

99

23

23

33

10

21

34

11

23

48

11

74

65

24

51

35

36

14

28

51

01

18

89

95

47

07

31

97

20

74

04

36

72

01

91

87

14

11

22

77

10

10

55

94

93

90

27

71

44

42

14

17

51

48

10

20

00

88

33

11

29

34

28

91

13

82

29

51

09

32

71

53

04

69

21

12

22

52

55

55

51

31

31

60

22

51

79

56

41

06

67

06

33

99

51

01

73

41

12

26

61

91

92

02

05

57

48

12

10

14

03

50

76

82

04

12

24

11

22

24

48

19

19

19

19

13

13

12

31

10

11

63

49

13

74

86

To

tal

42

81

28

45

11

02

12

42

48

63

12

67

57

51

37

13

76

46

46

17

74

06

79

20

36

98

93

02

5

P

pro

be

L

loci

P

ML

p

hy

sica

lly

map

ped

loci

P

ML

CA

p

hy

sica

lly

map

ped

loci

by

com

par

ativ

ean

alysi

sA

ctu

allo

cin

um

ber

may

be

incr

ease

d2

2

fold

Nucleic Acids Research 2004 Vol 32 No 12 3555

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

their precise location within the intervals was not knownThese 93 genes and 17 QTLs are colored green in Figure 1With an average of 36 the number of phenotypic markersvaried from 29 for group 4 to 44 for group 1 Comparisonof the genetic with the physical consensus maps localized 258of the 269 phenotypic markers and useful genes to 37 GRRs

The highest number (32) of phenotypic markers was observedfor the lsquo1S08rsquo region The 11 GRRs that lacked phenotypicmarkers were present in the proximal regions of the chromo-somes (Figure 1 and Table 1) The genes mapping in the non-GRR regions are Per1 Hk1 Lr35 Xksu905 (Wip) Lr25 Lr9Pm7 Lr30 Xwsu4 (Dor4) tav1933 (Vdoc) and v1

3556 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

Distribution of recombination

Comparison of the consensus physical and genetic linkagemaps via 428 common markers provided a detailed andaccurate estimate of recombination at a sub-regional level(Figure 1) In general a severe suppression of recombinationwas observed in the centromeric regions of the wheat

chromosomes (Figure 1) One-fourth of the wheat genomepresent around the centromeres accounted for lt1 of thetotal recombination Nearly perfect linkage was observedamong markers present on different arms of a chromosomeFor example markers Xcdo618 (lsquo1S04rsquo) and Xcdo98(lsquo1L02rsquo) were physically separated by one-fourth ofthe chromosomal length but were lt2 cM apart Due to the

Nucleic Acids Research 2004 Vol 32 No 12 3557

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

lack of recombination in the proximal regions a large num-ber of markers clustered in the centromeric regions of thelinkage maps (Figure 1)

The gene-poor regions accounted for only 5 of therecombination as 95 of the recombination was observedin the GRRs (Figure 1 and Table 1) Markers present intwo different GRRs separated by a gene-poor region were

usually tightly linked on the genetic linkage map For exam-ple no recombination was observed between markers Xwg789(lsquo1S06rsquo) and Xcdo618 (lsquo1S04rsquo) Similarly markers XksuD22(lsquo2L08rsquo) and Xcdo373 (lsquo2L10rsquo) were inseparable on thegenetic linkage map even though these were physically separ-ated by 20 of the armrsquos length (Figure 1) Maximumrecombination observed in a gene-poor region was for the

3558 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

region between lsquo4S09rsquo and lsquo4S07rsquo that accounted for 12of the armrsquos recombination Recombination occurring in theGRRs varied from 90 for group 4 to 985 for groups 6 and7 The chromosome 3L GRRs accounted for almost 100 ofthe armrsquos recombination Among GRRs maximum geneticlength of 143 cM was observed for lsquo3L09rsquo and minimumof 1 cM was for lsquo1S04rsquo region (Figure 1 and Table 1) Recom-bination rate for two GRRs (lsquo7L01rsquo and lsquo7L10rsquo) present on

the same chromosome arm was 53-fold different Only 1ndash3of the armrsquos recombination was observed for the GRRslsquo1L02rsquo and lsquo7L01rsquo (Figure 1)

Recombination in the distal chromosomal regions was muchhigher as compared to the proximal halves The recombinationrates among GRRs present in the distal half of the chromo-somes were however highly variable Recombination rates insome proximal GRRs were higher than in distal GRRs For

Nucleic Acids Research 2004 Vol 32 No 12 3559

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

example lsquo1L09rsquo region is proximal to lsquo1L10rsquo but is about5-fold higher in recombination (Figure 1) There are six otherexamples where recombination in the proximal GRRs is 38ndash325 higher compared to the distal GRRs (Figure 1)

In general the recombination rate in the smaller GRRsseemed higher as compared to the GRRs that encompassedlarger chromosomal regions The 10 Mb sized region

lsquo6S10rsquo had the highest percentage recombination per chromo-some arm (Table 1) Similarly the lsquo1S08rsquo region showed 86of the armrsquos recombination and is only 7 Mb in size On thecontrary a larger GRR lsquo4L05rsquo is 70 Mb but accounted forlt15 of the armrsquos recombination (Figure 1) As in the case oflsquo3L09rsquo this pattern of negative correlation between GRR sizeand recombination rate was not observed for some regions

3560 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

Comparisons of consensus physical and genetic linkagemaps showed that the ratio of physical to genetic distance(kbcM) varied as much as 140-fold among GRRs ThekbcM estimate ranged from 151 for lsquo1S08rsquo to 21687 forlsquo7S02rsquo with an average of 3381 (Figure 1 and Table 1) Inmost cases recombination rate was higher for GRRs with a

higher gene density although there were some exceptions Forexample gene density was relatively high in the GRR lsquo1S04rsquo(1 gene per 273 kb) but only 3 of the armrsquos recombination wasobserved in this region (Table 1) Similarly lsquo5S09rsquo region isrelatively low in gene density (1 gene per 2688 kb) but 60 ofthe armrsquos recombination occurred in this region

Nucleic Acids Research 2004 Vol 32 No 12 3561

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

Figure 1 (Group 1 to 7) Distribution of genes and recombination on wheat chromosomes Sizes of consensus chromosomes location and sizes of GRRs are drawn toscale based on the average size of the three homoeologous chromosomes for each homoeologous group (54) Names of the GRRs are given on the left side of aconsensus chromosome In the nomenclature of GRRs (eg lsquo1S08rsquo) the first digit represents wheat homoeologous group followed by the arm location either as shortarm (S) or long arm (L) The last two numeral numbers represent GRR location as fraction length (FL) of the chromosome (eg 08 for lsquo1S08rsquo) The flanking deletionlines for each GRR are shown in blue on the left side of a consensus physical chromosome Actual physical size (black) and the ratio of physical to genetic distance(red) for a region are given on the right hand side of a consensus chromosome Sizes (in Mb) of GRRs are calculated based on the cytological measurements(measurements of the region bracketed by the flanking deletion line breakpoints in comparison to the total chromosome size) and are drawn to scale Recombinationin a chromosomal region is calculated by comparing deletion line based physical map with the consensus genetic linkage map for each chromosome Flankingmarkers for each GRR are joined with dotted lines connecting physical and consensus genetic linkage maps On the consensus genetic linkage maps the genes forwhich exact location in reference to other markers was known are shown in red and the genesQTLs for which the location was imprecise are shown in green

fraction length for satellite F region not precisely marked by deletion lines C markers which were not flanked by the flanking deletion lines for the GRR butprobably are part of that region

3562 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

DISCUSSION

Gene-containing regions of wheat

The number of genes in most of the higher plants is expected tobe similar although variations due to ploidy changes areexpected The in silico approach based gene number estimatefor Arabidopsis is 25 000 and for rice ranges from 32 000 to50 000 (813) Accounting for polyploidy the correspondingnumber for wheat may be between 75 000 and 150 000 Usingthe average gene size of 25 kb that is found in rice only 12ndash24 (188ndash375 Mb) of the wheat genome is therefore expectedto contain genes The predicted number of genes in thesequenced eukaryotes dropped 2- to 3-fold with the availabil-ity of better computational and biological tools The humangenome once thought to have more than 100 000 genes con-tains 30 000 genes based on new stringent criteria andimproved gene-prediction programs Similarly the gene-con-taining fraction of the wheat genome may even be smaller thanthe above predictions Therefore precise localization anddemarcation of the gene-containing regions is particularlyimportant for wheat

Use of deletion lines to reveal the physical location of genemarkers and the approach to combine the mapping data of thethree homoeologs to generate a consensus map have beenshown previously (91026) Relative gene marker locationwas conserved among the three homoeologs even at the cur-rent density (Supplementary Figures AndashG) It was thereforepossible to construct consensus maps for the seven homoeo-logous groups The consensus maps had an average of 48 dele-tion breakpoints per chromosome group with a range from 40for group 3 to 58 for group 7 Based on the average chromo-some size of the homoeologs deletion breakpoints occurredevery 133 Mb for group 7 to 204 Mb for group 3 The break-points were two times more frequent around the GRRs thanrandomly expected once every 8 Mb Of the 334 deletions210 (63) occurred in the GRRs that encompassed 29 ofthe wheat genome (Table 1)

The distribution of genes on wheat chromosomes is strikingWheat chromosomes have regions of high gene density inter-spersed by vast expanses of unpopulated regions consisting ofrepeated DNA Based on the sample of 3025 gene loci 29 ofthe wheat genome contained 94 of the genes (Figure 1) Of the48 GRRs that were identified 18 were major spanning 11 ofthe genome but accounted for 60 of the genes All majorGRRs were present in the distal 35 of the chromosomes Thelong arms of wheat contained twice as many genes as the shortarms Of the total 94 genes present in GRRs 59 were presentin the 27 GRRs on the long arm and remaining in the 21 GRRs onthe short arms No significant correlation was observed betweenthe chromosome size and the gene fraction or the size of theGRRs For example group 3 has the largest chromosomesamong the wheat groups but contained only 13 of thewheat genes compared with group 5 chromosomes that con-tained 20 Roughly the gene fraction seemed to correlatewith the size of the GRRs The size of chromosomes encom-passing the GRRs is the largest for group 5 and the smallest forgroup 6 Correspondingly the gene fraction was the highest forgroup 5 and the lowest for group 6

The actual size of the GRRs may be smaller than that shownin Figure 1 because the accuracy of bracketing the GRRsis dependent upon the number of deletion breakpoints If

randomly occurring breakpoints of the 334 deletion linesare expected to occur every 16 Mb on the consensus physicalmap As mentioned earlier frequency of these chromosomalbreaks was about double in the GRRs Even with that fre-quency GRRs interspersed by lt8 Mb of gene-poor DNAwill not be resolved

As observed in wheat distribution of genes is uneven inother grass species as well In rice 20 of the genome hasnot been contigedsequenced in spite of extensive efforts (8)The unsequenced part of the genome is probably gene-poorAbout 21 of the sequenced portion of the rice genomecontains 40 of the genes (44) Translocation breakpoint-based physical maps showed that barley chromosomes arealso partitioned into gene-rich and gene-poor regions (45)Similarly in the smaller genome plant Arabidopsis genesare unevenly distributed on the chromosomes About 45of the genome accounts for about 25 000 genes with an averagegene size of 2 kb

Based on the expected size of the gene-containing fractiononly a small part of the currently demarcated GRRs is allegedto contain genes Assuming that the wheat genes may encom-pass only 188ndash375 Mb (12ndash24) of the genome the gene-containing fraction within the GRRs is predicted to be only12ndash24 The exact distribution of genes in the GRRs is notknown but preliminary results suggest that the GRRs mayfurther be partitioned into mini gene-rich and gene-poor com-partments Within the GRRs the estimated 75 000 to 150 000wheat genes are expected to occur every 10ndash20 kb Except forploidy differences the wheat and the barley genomes are verysimilar in gene synteny and composition An 11 Mbsequence for selected wheat and barley regions showed thatgene density within the GRRs may range from a gene every4ndash103 kb with an average of 10ndash20 kb (46ndash49) The size of thelsquogene-emptyrsquo blocks interspersed in these regions ranged from08 to 94 kb The largest contig among these wheat and barleyregions is 261 kb around the Mla locus (49) A 130 kb region inthe contig contained 23 genes with an average gene density ofa gene per 56 kb Size of the interspersing lsquogene-emptyrsquoregions ranged from a few hundred base pairs to 11 kbThe second region of high gene density was 40 kb longand contained 10 genes (a gene every 4 kb) The largestlsquogene-emptyrsquo region was 55 kb Another 60 kb barley contigspanned a 32 kb region around the bronze locus with a geneevery 32 kb (34) Very similar observations were made inother large genome grasses such as Triticum monococcumAegilops tauschii and maize (475051)

Localization of the gene containing regions of wheat isbased on studying gene distribution of 3025 gene loci bythree different methods (Table 2) Gene distribution by dele-tion-line-based physical mapping was very similar to thatrevealed by the comparative analysis between physical andconsensus genetic linkage maps Similarly distribution ofgene markers was comparable to that of phenotypically char-acterized genes Therefore we believe that the demarcationand distribution of genes given in Figure 1 is a good repres-entation of the wheat genome Since the current analysis wasbased on the study of 39ndash78 of the wheat genes relativenumbers among various GRRs may slightly vary with theanalysis of additional genes Considering the diversity andrandomness of the analyzed gene markers the size relativegene density and gene proportion are however not expected

Nucleic Acids Research 2004 Vol 32 No 12 3563

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

to change dramatically A probable exception might bemulticopy gene families that were not represented in thisstudy because of the technical difficulties The currentinterpretations will not hold true for the multicopy genes iftheir distribution is dramatically different from that of thesingle-copy genes

The presence of 6 of the genes in the gene-poor regionssuggests that additional yet smaller GRRs may exist that werenot identified during the current analysis Some genes wereobserved in the highly heterochromatic gene-poor regionsTwo cDNA clones mapped in the centromeric region ofwheat homoelogous group 1 chromosomes that is highly het-erochromatic and gene-poor Other such examples are genemarkers Xbcd1124 Xcdo1340 and Xpsr596 on chromosome1B Xpsr107 XunlBF483292 Xabg356 and Xbcd260 on group2 Xtam12 Xcdo681 Xbcd1278 Xbcd102 XksuI32 XksuF34Xksu609 Xcdo920 and Xwg222 on group 3 and Xbcd1092Xbcd734 Xcdo1337 and Xbcd1262 on group 4

Distribution of recombination

Recombination is highly uneven along the wheat chromo-somes (Figure 1) Recombination mainly occurred in the48 GRRs that accounted for 95 of the recombinationMore than 100-fold differences were however observed forthe rate of recombination among various GRRs A part of thisdifference seemed to be due to the suppression effect of cen-tromere on recombination Essentially no recombination wasobserved in the proximal 30 of the wheat chromosomesin spite of the presence of GRRs In group 7 chromosomeseg genetic distance between the GRRs lsquo7S02rsquo andlsquo7L08rsquo is only 7 cM The spanned region is gt50 of thechromosome and extended over three GRRs Most of thehighly recombinogenic GRRs were localized in the distalparts of the chromosomes but not all distal GRRs were highlyrecombinogenic Genetic distances spanned by lsquo2L08rsquo andlsquo6L07rsquo GRRs were lt5 cM

Recombination distribution within the GRRs is not knownbut the available data suggest that recombination is highlyasymmetrical even within the highly recombinogenicGRRs Analysis of a 1 Mb region of barley homoeologousto one of the most recombinogenic region of wheat (lsquo1S08rsquo)identified a 240 kb region spanning the Mla locus where norecombination was observed (32) Recombination differencesup to 10-fold were observed among different sectors of theremaining part of the region Similar observations have alsobeen made in other eukaryotes Distribution of recombinationhas been best studied on yeast chromosomes Measured at 1 kbresolution the average frequency of recombination was035 cMkb with a range of 0ndash2 cMkb (httpwwwyeast-genomeorg) Measured at a 150 kb interval recombinationwithin a 1 Mb region of rice ranged from 0 to 15cM eventhough the gene distribution was fairly even (44) (httprgpdnaaffrcgojpPublicdatahtml) The average frequencyof recombination in rice is 0003 cMkb with a range of0ndash006 cMkb (httprgpdnaaffrcgojpPublicdatahtml)Similarly in wheat at a resolution of 7 Mb the averagerecombination frequency was 00003 cMkb with a rangefrom 0 to 0007 cMkb Non-recombinogenic regions havebeen observed in yeast as well as in rice but the highestrate of recombination for a region appears to be 35-fold

less in rice It may be because the rice estimates were at150 kb resolution whereas the recombination hotspotsmay be much smaller in length In yeast there is a majorhotspot around PHO8 gene located on the distal end of chro-mosome IV where recombination is 2 cMkb (httpwwwyeastgenomeorg) A 194 bp region around the waxy(wx) locus of rice has a recombination rate of 006 cMkb thatis 20-fold higher than the genome average (52) Similarly a377 bp region of the maize genome has a recombination rate of002 cMkb that is about 30 times higher than the genomeaverage (3553)

SUPPLEMENTARY MATERIAL

Supplementary Material is available at NAR Online

ACKNOWLEDGEMENTS

A contribution of the Agriculture Research Center WashingtonState University Pullman WA Journal series No 0902-03This research project was supported by the US Department ofAgriculture-National Research Initiative (USDA-NRI) and theWSU Vogel Endowment Funds

REFERENCES

1 SearsER (1954) The aneuploids of common wheat Research Bulletin572 University of Missouri Agricultural Experiment Station pp 1ndash58

2 ArumuganathanK and EarleED (1991) Estimation of nuclear DNAcontent of plants by flow cytometry Plant Mol Biol Rep 9 229ndash241

3 FlavellRB BennettMD SmithJB and SmithDB (1974) Genomesize and the proportion of repeated nucleotide sequence DNA in plantsBiochem Genet 12 257ndash269

4 SandhuD and GillKS (2002) Gene-containing regions of wheat and theother grass genomes Plant Physiol 128 803ndash811

5 SidhuD and GillKS (2004) Distribution of genes and recombination inwheat and other eukaryotes Plant Cell Tissue Organ Cult in press

6 ClaytonWD (1981) Evolution and distribution of grassesAnn Missouri Bot Gard 69 5ndash14

7 KelloggE (1998) Relationships of cereal crops and other grassesProc Natl Acad Sci USA 95 2005ndash2010

8 GoffSA RickeD LanTH PrestingG WangR DunnMGlazebrookJ SessionsA OellerP VarmaH et al (2002) A draftsequence of the rice genome (Oryza sativa L ssp japonica) Science 29692ndash100

9 GillKS GillBS EndoTR and BoykoE (1996) Identification andhigh-density mapping of gene-rich regions in chromosome group 5of wheat Genetics 143 1001ndash1012

10 SandhuD ChampouxJA BondarevaSN and GillKS (2001)Identification and physical localization of useful genes and markers to amajor gene-rich region on wheat group 1S chromosomes Genetics 1571735ndash1747

11 AkhunovED GoodyearAW GengS QiLL EchalierB GillBSMiftahudin GustafsonJP LazoG ChaoS et al (2003) Theorganization and rate of evolution of wheat genomes are correlatedwith recombination rates along chromosome arms Genome Res13 753ndash763

12 BarakatACarelsN and BernardiG (1997) The distribution of genes inthe genome of Gramineae Proc Natl Acad Sci USA 94 6857ndash6861

13 TAGI (2000) Analysis of the genome sequence of the floweringplant Arabidopsis thaliana Nature 408 796ndash815

14 BarakatA HanDT BenslimaneA RodeA and BernardiG (1999)The gene distribution in the genomes of pea tomato and date palmFEBS Lett 463 139ndash142

3564 Nucleic Acids Research 2004 Vol 32 No 12

15 SumnerAT TorreJDL and StuppiaL (1993) The distribution ofgenes on chromosomes A cytological approach J Mol Evol 37117ndash122

16 ClayO and BernardiG (2001) The isochores in human chromosome 21and 22 Biochem Biophys Res Commun 285 855ndash856

17 JabbariK and BernardiG (2000) The distribution of genes in drosophilagenome Gene 18 1859ndash1867

18 LanderES LintonLM BirrenB NusbaumC ZodyMCBaldwinJ DevonK DewarK DoyleM FitzHughW et al (2001)Initial sequencing and analysis of the human genome Nature 409860ndash921

19 McPhersonJD MarraM HillierL WaterstonRH ChinwallaAWallisJ SekhonM WylieK MardisER WilsonRK et al (2001)A physical map of the human genome Nature 409 934ndash941

20 VenterJC AdamsMD MyersEW LiPW MuralRJSuttonGG SmithHO YandellM EvansCA HoltRA et al(2001) The sequence of the human genome Science 291 1304ndash1351

21 GillKS GillBS EndoTR and TaylorT (1996) Identificationand high-density mapping of gene-rich regions in chromosome group 1of wheat Genetics 144 1883ndash1891

22 SandhuD and GillKS (2002) Structural and functional organization ofthe lsquo1S08 gene-rich regionrsquo in the Triticeae Plant Mol Biol 48791ndash804

23 DvorakJ and ChenK-C (1984) Distribution of nonstructural variationbetween wheat cultivars along chromosome arm 6Bp evidence from thelinkage map and physical map of the arm Genetics 106 325ndash333

24 CurtisCA and LukaszewskiAJ (1991) Genetic linkage betweenC-bands and storage protein genes in chromosome 1B of tetraploid wheatTheor Appl Genet 81 245ndash252

25 WernerJE KotaRS and GillBS (1992) Distribution of telomericrepeats and their role in the healing of broken chromosome ends in wheatGenome 35 844ndash848

26 GillKS GillBS and EndoTR (1993) A chromosome region-specificmapping strategy reveals gene-rich telometric ends in wheatChromosoma 102 374ndash381

27 KotaRS GillKS GillBS and EndoTR (1993) A cytogeneticallybased physical map of chromosome 1B in common wheat Genome36 548ndash554

28 FarisJD HaenKM and GillBS (2000) Saturation mapping of agene-rich recombination hot spot region in wheat Genetics 154823ndash835

29 WengY TuleenNA and HartGE (2000) Extended physical maps anda consensus physical map of the homoeologous group-6 chromosomesof wheat (Triticum aestivum L em Thell) Theor Appl Genet 100519ndash527

30 TanksleySD GanalMW PrinceJP de VicenteMCBonierbaleMW BrounP FultonTM GiovannoniJJ GrandilloSMartinGB et al (1992) High density molecular linkage maps of thetomato and potato genomes Genetics 132 1141ndash1160

31 PuechbertyJ LaurentAM GimenezS BillaultA LaurentMEBCalendaA MarcaisB PradesC LoannouP YurovY et al (1999)Genetic and physical analyses of the centromeric and pericentromericregions of human chromosome 5 Recombination across 5cen Genomics56 274ndash287

32 WeiF Gobelman-WernerK MorrollSM KurthJ MaoLWingRA LeisterD Schulze-LefertP and WiseRP (1999)The Mla (powdery mildew) resistance cluster is associated withthree NBS-LRR gene families and suppressed recombination withina 240-kb DNA interval on chromosome 5S (1HS) of barley Genetics153 1929ndash1948

33 FeuilletC and KellerB (2002) Comparative genomics in the grassfamily molecular characterization of grass genome structure andevolution Ann Bot (Lond) 89 3ndash10

34 FuH ZhengZ and DoonerHK (2002) Recombination rates betweenadjacent genic and retrotransposon regions in maize vary by 2 ordersof magnitude Proc Natl Acad Sci USA 99 1082ndash1087

35 YaoH ZhouQ LiJ SmithH YandeauM NikolauBJ andSchnablePS (2002) Molecular characterization of meiotic

recombination across the 140-kb multigenic a1-sh2 interval of maizeProc Natl Acad Sci USA 99 6157ndash6162

36 AdamsMD CelnikerSE HoltRA EvansCA GocayneJDAmanatidesPG SchererSE LiPW HoskinsRA GalleRF et al(2000) The genome sequence of Drosophila melanogaster Science 2872185ndash2195

37 BoykoEV GillKS Mickelson-YoungL NasudaS RouppWJZiegleJN SinghS HassawiDS FritzAK NamuthD et al(1999) A high-density genetic linkage map of Aegilops tauschiithe D-genome progenitor of bread wheat Theor Appl Genet 9916ndash26

38 EndoTR (1982) Gametocidal chromosomes of three Aegilops speciesin common wheat Can J Genet Cytol 24 201ndash206

39 EndoTR MukaiY YamamotoM and GillBS (1991) Physicalmapping of a male-fertility gene of common wheat Jpn J Genet66 291ndash295

40 EndoTR and GillBS (1996) The deletion stocks of common wheatJ Hered 87 295ndash307

41 AndersonJA OgiharaY SorrellsME and TanksleySD (1992)Development of a chromosomal arm map for wheat based on RFLPmarkers Theor Appl Genet 83 1035ndash1043

42 SandhuD SidhuD and GillKS (2002) Identification of expressedsequence markers for a major gene-rich region of wheat chromosomegroup 1 using RNA fingerprinting-differential display Crop Sci 421285ndash1290

43 DilbirligiM EraymanM SandhuD SidhuD and GillKS (2004)Identification of wheat chromosomal regions containing expressedresistance genes Genetics 166 461ndash481

44 WuJ MaeharaT ShimokawaT YamamotoS HaradaCTakazakiY OnoN MukaiY KoikeK YazakiJ et al (2002) Acomprehensive rice transcript map containing 6591 expressed sequencetag sites Plant Cell 14 525ndash535

45 KunzelG KorzumL and MeisterA (2000) Cytologically integratedphysical restriction fragment length polymorphism maps for thebarley genome based on translocation breakpoints Genetics 154397ndash412

46 ShirasuK SchulmanAH LahayeT and Schulze-LefertP (2000) Acontiguous 66-kb barley DNA sequence provides evidence for reversiblegenome expansion Genome Res 10 908ndash915

47 WickerT SteinN AlbarL FeuilletC SchlagenhaufE and KellerB(2001) Analysis of a contiguous 211 kb sequence in diploid wheat(Triticum monococcum L) reveals multiple mechanisms of genomeevolution Plant J 26 307ndash316

48 BrueggemanR RostoksN KudrnaD KilianA HanF ChenJDrukaA SteffensonB and KleinhofsA (2002) The barley stem rust-resistance gene Rpg1 is a novel disease-resistance gene with homology toreceptor kinases Proc Natl Acad Sci USA 99 9328ndash9333

49 WeiF WingRA and WiseRP (2002) Genome dynamics andevolution of the Mla (powdery mildew) resistance locus in barley PlantCell 14 1903ndash1917

50 FeuilletC and KellerB (1999) High gene density is conserved atsyntenic loci of small and large grass genomes Proc Natl Acad SciUSA 96 8265ndash8270

51 TikhonovAP SanMiguelPJ NakajimaY GorensteinNMBennetzenJL and AvramovaZ (1999) Colinearity and its exceptions inorthologous adh regions of maize and sorghum Proc Natl Acad SciUSA 96 7409ndash7414

52 InukaiT SakoA HiranoHY and SanoY (2000) Analysis ofintragenic recombination at wx in rice Correlation between the molecularand genetic maps within the locus Genome 43 589ndash596

53 CivardiL XiaY EdwardsKJ SchnablePS and NikolauBJ (1994)The relationship between genetic and physical distances in thecloned a1-sh2 interval of the Zea mays L genome Proc Natl Acad SciUSA 91 8268ndash8272

54 GillBS FriebeB and EndoTR (1991) Standard karyotype andnomenclature system for description of chromosome bands andstructural aberrations in wheat (Triticum aestivum) Genome 34830ndash839

Nucleic Acids Research 2004 Vol 32 No 12 3565