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Progress on the sequencing of theeuchromatic gene rich space ofchromosome 6 of Solanum lycopersicumcv. Heinz 1706
Sander Peters
Cologne Oct 2008
Outline
►Sequencing & assembly status
►Euchromatin and heterochromatin boundaries
►Integrated Map
►Physical gaps
►Gene and repeat content
►Conclusions
FISH, sequencing and BAC walking progress status
► 155 candidate BACs| 106 Hind, 34 Mbo, 15 Eco
► 113 BACs FISHed on Cherry VNFT and Heinz| 81 BACs on VI
| 3 on VI and other
| 28 on other
| 1 no signal
► 143 BACs Physically mapped| 64 seed BACs
• 59 Hind, 5 Mbo| 79 extension BACs
• 39 Hind, 15 Eco, 25 Mbo
► Sequencing & assembly| 47 BACs phase 3
| 96 BACs ordered contigs
| Average overlap 13.3 kb, 100% match
| AGP and TPF @ SGN
► 10.7 Mb| 2.0 Mb short arm, 1.8 Mb pericentromere, 6.9 Mb long arm
► 157 markers identified (31% not mapped on chr 6)
0
10
20
30
40
50
60
70
80
contig size (nts)#
cont
igs
0
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
supercontigsize (#BACs)
#s
up
erc
on
tig
s
Borders of 6E and 6H
H060A01
H315H13
H310B09H309D09
L. Telo
S. TeloH016K14H158P14
H147H20S. Telo
6SE: 0cM-10cM
6SH+6LH: 10cM-18.5cM
6LE: 18.5cM-101cM
Integrated map
► Integrated map
| Crosslink between 157 genetic markers, 10.7 Mb of sequence and (cyto)genetic locations of 81 BACs on tomato chromosome 6
| 31% markers not mapped on EXPEN 2000
| Gaps on 6LE
• 73 cM (T0405) and 93 cM (C2_At1g18640)
H060A01
M082G10
H012O10
H167M06
M012J12
H261A18
H034C13
H215M16
H169D11
H194N16
H301C21
H052N09
H098L02
H060A01
M012J12
H182D16
H055E14
H309D09
H024F02
H046G10
H052N09
H060A01H315H13
H0307J13
H066I09
H021K07
Gaps between BAC supercontigs
TG115
TG482 cLEY-13-J2
H098L02
H021K07
H028D14
E005I21
M066F23
TG193
H315H13
TG215TG314
U1461
40
E014B21 M059G17
T1789
TG642
H057D21C2_At1
g1687
0
M106A20
H060A01 H182D16
H034C13
T0123T1090
cTOE-1
1-C4
SSR122
T1785
T1124
T1449
C2_At2
g2849
0
M012J12
C2_At1g18640
H169D11
TG579
E131F12
T0405
H055E14
H203N09 M099A03
H116G14
C2_At4
g2110
5T01
40
H059D21
H038F03
U144275
T0585T0798
E037L07
T1066
H084A18
M113N10
H304I22
H117B06
H246E15
T1272cLEY-15-N18 T1835
T0447
C2_At1
g2005
0
GAP
Cytogenetic map order Genetic map order BAC Tiling Path
Determination of gap sizes distances
►Pachytene complements
| measurements of physical distances on a micrometer scale
• ‘global’
• ‘local’ between adjacent BAC FISH positions
single focus
147H2052O17107A05251G05250I21304P16134P07242H1920O1726P1823B17
(0.1)
(1.29)1.051.22
4.2713.06
0.2
2.48
Multi-color BAC FISH on pachytene
Physical gaps in integrated map
► Pachytene complements
| measurements of physical distances on a micrometer scale
• ‘global’ and ‘local’ between adjacent BAC FISH positions
| Estimation of base pair / m / cM relationship
• Large gap on the genetic map between 73 and 93 cM
• 0.62 Mb/m
• 1.2 Mb
domain chr. distance m)
gen.distance (cM)
Size (Mb)
Mb/m cM/m Mb/cM
6SE 1.8 10 4.1 2.27 5.5 0.41
6SH+6LH 8 8.5 50 6.25 1.1 5.94
6LE 29.5 82.5 26.9 0.91 2.8 0.33
Gene and repeat content
Conclusions
► Megabase-sized gaps which are not poorly targeted with markers and for which we cannot obtain anchored BACs (BAC-walking?)
► Complement sequencing effort using NGS platforms
► However, whole genome assembly (WGA) is a challenge
► Sequence islands may serve as backbone for WGA
► We need more anchored BACs! BAC FISH and BAC sequencing will remain indispensable.
► Sequence comparison between tomato and BACs from other Solanum species combined with cross-species multicolor BAC-FISH painting may allow identification of new candidate seed BACs.
Applied Bioinformatics
Elio Schijlen
Marjo van Staveren
Jan van Haarst
Marleen Henkens
Roeland van Ham
Bas te Lintel Hekkert
Joost de Groot
Thamara Hesselink
Laboratory of Genetics
Dóra Szinay
Hans de Jong
Ludmilla Khrustaleva
Song-Bin Chang
Xiaobo Zhong
Willem Stiekema
René Klein Lankhorst
Erwin Datema
José van de Belt
Sander Peters
single focus
Euchromatin and heterochromatinbordering BACs on chromosome 6
H060A01H020O17
H091L20
H112G05
H021K07
H271L05
S. Telo
H060A01
H315H13
H310B09
H309D09
L. Telo
cen
H091L20
H003K02 10 TG178
H020O17 CT135*
H060A01 101 Ct_At1g20050
H315H13 98 TG193L. Telo
BAC cM Marker
H016K14 32
H158P14 2 T0686
H147H20 0 CT216
H107A05 3 T1188
H112G05 5.5 Mi
S. Telo
H54K13
H147H20
H176K10
S. TeloH016K14
H158P14
H147H20S. Telo
H107A05
H147H20S. Telo
single focus
Chr. 6 pericentromeric & subtelomeric BACs
H003K02
Cen
S.Telo
040F08091L20
282A22
H032D19
S.Telo
Cen
L.Telo
H309K01
H242H19S.Telo ?
H295L11
Cen
L.Telo
H309K01 10 cLET- A.thal. conserved 5-A4 centromere sequence chr. 5
H003K02 10 TG178 A.thal. copia like transposon
H242H19 12 T1063 A.thal. chr. 1 centr. linked repeat
H295L11 10 T0244 A. thal. chr. 4 copia like transp.
H040F08 12 CD67 A. thal. Ty3 like transposon
BAC cM Marker BLAST
H016K14 32 none TTTAGGG ?
S. Telo
cen
L. Telo
H315H13 98 TG193
TR
Extended DNA fibre of distal end chromosome arm 6S
TGR1+
+
--