Microsatellite markers from a microdissectedswine chromosome 6 genomic libraryF Zhao, S Ambady, F A Ponce de LeoÂn, L M Miller, J K Lunney, D R Grimm,L B Schook, C F Louis

Abstract

To develop additional microsatellite (MS) mar-

kers in the region of the porcine skeletal muscle

ryanodine receptor gene (RYR1), a microdis-

sected genomic library was generated from the

proximal half of the q arm of swine chromosome

6. Purified DNA was restriction enzyme-

digested, ligated to oligonucleotide adaptors

and amplified by PCR using primers comple-

mentary to the adaptor sequences. The purity of

the amplified products and boundaries of the

microdissected chromosomal region were ver-

ified by fluorescence in situ hybridization.

(CA)n-containing sequences were then identi-

fied in a small insert genomic library generated

from the PCR-amplified microdissected DNA.

Oligonucleotide primers were developed for the

PCR amplification of 30 of the 46 (CA)n repeat-

containing clones, which were subsequently

used to amplify DNA isolated from unrelated

pigs of different breeds to determine the

informativeness of these MS markers. Twenty-

two of these MS markers were genotyped on the

University of Illinois Yorkshire ´ Meishan

swine reference population. These 22 markers

were all assigned within a 50.7-cm region of the

swine chromosome 6 linkage map, indicating

the specificity of the microdissected library.

Keywords: swine, microsatellite markers, chro-

mosome microdissection, linkage map

Introduction

To more precisely map the genes that are

responsible for specific traits of interest in the

pig, several laboratories have sought to improve

the resolution of swine genetic linkage maps

(Rohrer et al. 1994, 1996; Archibald et al. 1995;

Ellegren et al. 1994). The current study has

focused on swine chromosome 6, which con-

tains the RYR1 gene that has been shown to be

responsible for porcine stress syndrome (PSS)

in pigs (Fuji et al. 1991). PSS is known to be

associated with a decreased fat content and

increased muscling (Rempel et al. 1995). While

it has been proposed that the altered skeletal

muscle calcium regulation of the T allele of the

RYR1 gene might be responsible for the

increased muscling in pigs (MacLennan &

Phillips 1992), it is still not clear how a

mutation in this protein could produce the

beneficial carcass traits associated with PSS

pigs. The alternative hypothesis is that the

positive carcass traits of the RYR1 T/T animals

are due to allelic variants of a gene that is close

to but distinct from the RYR1 locus on pig

chromosome 6. We have previously prepared

small insert genomic libraries from swine

chromosome 6 DNA isolated by whole chromo-

some micro-isolation (Ambady et al. 1997) or

bivariate flow sorting (Grimm et al. 1997).

However, these libraries only produced six

new MS markers in the 40 cm region flanking

the RYR1 gene, furthermore, MS markers fre-

quently lose their informativeness when

mapped in inbred populations (Paszek et al.

1998). To address this issue, we isolated MS

markers from a small insert genomic library

prepared from DNA that had been microdis-

sected from the proximal half of the q arms of

swine chromosome 6.

Materials and methods

Chromosome microdissection and library con-

struction

Briefly, swine chromosome 6 was identified

under a microscope and one half of the q arm of

the chromosome was scraped and collected

from 15 metaphase spreads. The purified DNA

was then used to construct a genomic DNA

library in Lambda ZAP Expression vector using

Gigapack II Gold packing extract (Stratagene,

San Diego, CA) and screened with a 32P-labeled

(GT)10 probe as described by Ponce de LeoÂn et

al. (1996) and Ambady et al. (1997). Individual

positive phage clones were eluted and subjected

to PCR amplification using T3 and T7 oligonu-

cleotide primers as described by Ambady et al.

Animal Genetics,

1999, 30, 251±255

F ZhaoC F Louis*Department of Biochem-istry, University of Min-

nesota, Minneapolis,

MN 55455, USA

*Present address:Department of Biochem-

istry, University of Min-

nesota, 435 Delaware

Street, S.E. Minneapolis,MN 55455, USA

S AmbadyF A Ponce de LeoÂnDepartment of Animal

Science, University of

Minnesota, St. Paul,

MN 55108, USAL M MillerL B SchookDepartment of Veterin-

ary PathoBiology, Uni-versity of Minnesota,

St. Paul, MN 55108,

USAJ K LunneyD R GrimmU S D A - A R S - I D R L ,

BARC-East, Building1040, Room 105, Belts-

ville, MD 20705, USA

Correspondence: Dr Charles F Louis.

Accepted 5 January 1999

ã 1999 International Society for Animal Genetics 251

(1997). PCR-amplified DNA products were

purified using Qiagen PCR purification columns

(Qiagen, Inc. Santa Clarita, CA) and used as

templates for dye-terminator fluorescence cycle

sequencing (ABI). Sequences were checked for

uniqueness with respect to the USDA-MARC

database (Dr Gary Rohrer, personal communica-

tion) and for the presence of porcine repetitive

elements (Alexander et al. 1995). Oligonucleo-

tide PCR primers for MS sequences were

designed using the PRIMER program (Lincoln

et al. 1991). Fluorescence in situ hybridization

was performed as described in Ambady et al.

(1997).

Genotyping and linkage analysis

Genotyping of MS markers was performed as

described by Ambady et al. (1997). Segregation

of these MS markers was analyzed with

CRIMAP version 2.4 (Green et al. 1990).

Results

Chromosome microdissection

Swine chromosome DNA was isolated from the

region encompassing the RYR1 gene by micro-

dissecting approximately half of the proximal

region of chromosome 6q from 15 metaphase

spreads of swine lymphocytes. PCR amplifica-

tion of the microdissected chromosomal DNA

resulted in the production of DNA fragments

that ranged from 200 to 1500 bp (data not

shown). A FISH paint probe was subsequently

prepared to verify the chromosomal specificity

and the physical boundaries of the microdis-

sected DNA. Fluorescence signals were only

observed on the proximal region of the chromo-

some 6 q arm encompassing the RYR1 gene,

which is located at 6q11±21 (Harbitz et al. 1990)

(data not shown).

The Lambda ZAP DNA library had 1.2 ´ 106

pfu with 95% recombinant clones. Microsatel-

lite markers were identified by screening this

genomic DNA library with a 32P labeled (GT)10

oligonucleotide probe. Over 300 clones were

identified from the initial screening of 30 000

clones in this genomic library. Eighty-five of

these clones were selected and subjected to

secondary screening, of which 67 clones still

hybridized to the (GT)10 oligonucleotide probe.

Forty-six of these clones produced a discrete

DNA band on agarose gels after a third screen

and were used as templates for dye-terminator

fluorescence cycle sequencing.

DNA sequence analysis revealed that all 46

clones contained (CA)n repeats, mostly of the

imperfect type with one or more interruptions

in the run of CA repeats (Weber 1990), with the

number of CA repeat units varying from 10 to

43. Of the 46 MS sequences, four were dupli-

cates and three were triplicates (leaving 36 MS

sequences). Sequence comparison with the

USDA-MARC database revealed that six of

these 36 MS sequences were identical to

previously identified MS, leaving 30 unique

MS sequences. The DNA sequences of the 30

unique clones were analyzed using the PRIMER

program and oligonucleotide primers were

designed based on the flanking sequences of

the (CA)n repeats. Oligonucleotide primer and

the optimized PCR annealing temperatures for

these MS markers, are listed in Table 1.

Polymorphism of the MS markers and their

placement on the pig genetic linkage map

To assess their heterozygosity, the 30 unique

MS markers were first tested on 24 unrelated

animals randomly selected from six breeds of

pigs (three from Meishan, seven from Yorkshire,

three from Duroc, three from Hampshire, three

from Landrance and five from Pietrain), includ-

ing the grandparent generation of the University

of Illinois Meishan ´ Yorkshire reference popu-

lation (Clamp et al. 1993). Twenty-nine of the

MS markers were found to be polymorphic

(Table 1). Twenty-two of these markers were

polymorphic in the University of Illinois refer-

ence population; the remaining markers were

monomorphic or had a low heterozygosity in

the reference population so could not be used in

linkage analysis in this study.

Linkage analysis was performed by genotyp-

ing the 22 polymorphic MS markers on a subset

of the reference population comprising 96

individuals from eight families selected for

their litter sizes and informativeness with

framework markers on chromosome 6 (Paszek

et al. 1995). The previously reported chromo-

some 6 framework markers (Paszek et al. 1995)

were incorporated into the analysis as anchor-

ing points. The analysis also included addi-

tional MS markers from the USDA-MARC

comprehensive swine genome map (Rohrer

et al. 1994, 1996) and recently identified MS

markers derived from micro-isolated and flow-

sorted chromosome 6 genomic libraries

(Ambady et al. 1997; Grimm et al. 1997; Hoy-

heim, personal communication).

Multipoint linkage analysis revealed that

these 22 MS markers were all clustered in a

50.7-cm region corresponding to the dissected

region on this chromosome. Their addition

resulted in a chromosome 6 linkage map

252

Zhao, Ambady,

Ponce de LeoÂn et al

ã 1999 International Society for Animal Genetics, Animal Genetics 30, 251±255

Table

1.

Ch

ara

cteri

zati

on

of

the

30

new

swin

em

icro

sate

llit

em

ark

ers

deri

ved

from

the

mic

rod

isse

cte

dch

rom

oso

me

6gen

om

icli

bra

ry

Mark

er

nam

eF

orw

ard

pri

mer

Revers

ep

rim

erA

nn

eal

ing

tem

p.

(°C

)

Nu

mber

of

all

ele

sa

All

ele

sizes

(bp

)aH

ete

rozygosi

tya

Map

ped

in

ref.

fam

ily

MN

10

CCAAGACCCAGCATCAGG

CGTGCAGACATGGAAACG

60

6114±126

0. 5

7Y

es

MN

11

GTGGTTTGGAGGATGCTTG

CTTGGGTAGATACCAAGGCC

55

7129±149

0. 6

3Y

es

MN

12

CTGTAATTCCTGTGGTTGG

TGATGGTGGACCGGCATAAG

55

5159±181

0. 6

7Y

es

MN

13

CTGAAACAGAGGCTTACTG

TCTGAGATGACTGGACGAC

55

3135±141

0. 3

0Y

es

MN

14

CCTATGGCAGGCAGCTCAG

AGCAGCTTGCGGCTCTC

60

4156±166

0. 2

1Y

es

MN

15

CAACAAATGCTGCGTGTTCC

CCATGTCACTTGTTGTTCTC

55

10

115±147

0. 5

8Y

es

MN

16

GGGTTAATGGATGAATGG

TCCATTATGTCTAGACAC

57

10

82±102

0. 5

3Y

es

MN

17

ATACCTTGCCAGTGAGGGAC

TGCTATGGTTGTGGTGAAGG

57

8131±155

0. 4

5Y

es

MN

18

GGGAGAGAGTCTGCAAGG

CTGTGCCCTCAGAGGAAG

60

4137±143

0. 1

7Y

es

MN

19

CTCCACTCATCATCAAC

GATGATGAGTTATGAGTGTG

55

4119±130

0. 4

3Y

es

MN

20

GATATGTGTCCCACAGATGC

CGGGAGATTTGAGGATG

57

6147±161

0. 5

8Y

es

MN

21

CTCCAGAATCTAAGCTCTC

AGGACTCCGTGAGAGTTCTC

60

14

137±193

0. 6

7Y

es

MN

22

ACAGGAAGTGTGGAAAGCC

CCAGAGTTCCCTGAATAC

55

3220±224

0. 2

5Y

es

MN

23

GATATCTGTAGACGTTGCC

ATGGTACTCAACCAGGT

55

11

110±148

0. 7

5Y

es

MN

24

ACCTGTGTTCTGTCACTCC

AAGTCTTCGGGGTCTGAGG

57

8121±135

0. 4

3Y

es

MN

25

CAGTGTTCTGCTACACAG

CTAGTGTCTTGGAGTGGAGCT

55

6137±145

0. 6

5Y

es

MN

26

AACCTCTACCCACTCTCTC

CCATGGGGAAATACTACAC

55

6136±145

0. 4

3Y

es

MN

27

GGTTTGGTGACTTGGTG

TATCTCTCACTTCCCTG

57

9230±252

0. 7

0Y

es

MN

28

GACAGAATGGACTATTGG

TCAGATGTGTGGAGTGG

55

7179±201

0. 4

3Y

es

MN

29

GTATCTTCTTGTTGCTGC

GAGTGTTCACTTGGTGG

57

8212±233

0. 7

5Y

es

MN

30

TGTGCAGCTTGCAGGAG

AGGAGCGGGAAATGTCTCTG

55

5157±169

0. 6

1Y

es

MN

31

GCTGCTGGATGTGTTAGAG

CACATGCGTGTGCACATG

60

3196±200

0. 3

8Y

es

MN

32

bTGTGCACAATCCTGAG

CTGTATCACTCACAGTGC

57

5126±138

0. 2

5N

o

MN

33

ACGTGGCTCTGGCTCTG

GGGAGCCAGGGAAGATG

60

3157±173

0. 3

8N

o

MN

34

CCATACATATGCCATGGG

GAACACAGTTTGGGTGGC

57

2211±219

0. 0

4N

o

MN

35

CACATCCCTTCATTCTCCG

GCATGTCACTTCTGCCTGC

57

2181±185

0. 0

4N

o

MN

36

TGTGTTAGTTTCAGGTAC

CTGTATAGCACAGGGAAC

55

2124±126

0. 2

2N

o

MN

37

CCCTCAGTAGAGCCATTATC

GCTGAGCAGCAATTGGCTG

57

6112±138

0. 4

6N

o

MN

38

CTCCATATGCCACCAGAG

AGTGGCCCCTACCTGTTG

60

1170

0. 0

0N

o

MN

39

GGAGCTGCTGACGTCTG

CCAGACAGAACAGAGATG

57

8155±199

0. 4

2N

o

aC

alcu

late

dfr

om

14

un

rela

ted

US

pig

san

dth

eF

1gen

era

tion

of

the

Un

iver

sity

of

Illi

nois

refe

ren

ce

pop

ula

tion

.bP

oly

morp

hic

,bu

td

iffi

cult

tosc

ore

inth

eU

niv

ersi

tyof

Illi

nois

refe

ren

ce

fam

ily.

253

Microsatellites from

microdissected swine

chromosome 6

ã 1999 International Society for Animal Genetics, Animal Genetics 30, 251±255

comprised of 70 MS markers with an average

density of one MS marker per 1.3 cm (Fig. 1).

The resulting sex-averaged length of the map is

207.7 cm based on the placement of MS markers

SW2535 and SW2052 at the ends of the p and q

arms, respectively.

Discussion

The data reported here demonstrate that chro-

mosome microdissection is an effective

approach for the development of additional

microsatellite markers from a defined region of

the porcine genome. The redundancy rate of

this library was 22% (10/46), which is a little

higher than the 12% for the bovine micro-

dissected chromosome 1 library (Sonstegard

et al. 1997) and the 15% for the previously

reported flow sorted chromosome 6 genomic

DNA library (Grimm et al. 1997). Linkage

Fig. 1. Cytogenetic and linkage map of swine chromosome 6. The linkage map was based on the University of

Illinois reference population. Markers in italic are from the previously reported chromosome 6 framework map

(Paszek et al. 1995); the linkage map also includes previously assigned chromosome 6 microsatellite markers

(Rohrer et al. 1994, 1996; Ambady et al. 1997; Grimm et al. 1997; Hoyheim, personal communication). The

linkage map on the right is an expanded view of the 50.7 cm interval where the new microsatellite markers

(MN10±MN31) derived from the microdissected chromosome 6 genomic library were mapped.

254

Zhao, Ambady,

Ponce de LeoÂn et al

ã 1999 International Society for Animal Genetics, Animal Genetics 30, 251±255

analysis suggested that many alternative mar-

ker orders were plausible, as indicated by

similar likelihood, because of the high density

of markers and their varying informativeness in

the reference population. Some of the new MS

markers reported here (MN11 and MN15; MN12

and MN26; MN19, MN20 and MN27) colocalize

on this map, so alternative mapping strategies

with greater resolving power, such as radiation

hybrid panels (Cox et al. 1990; Slonim et al.

1997) or large insert YAC or BAC libraries, will

be needed to determine the fine order of the

MS markers developed in this study along

with markers from previous maps (Rohrer

et al. 1994, 1996; Ambady et al. 1997; Grimm

et al. 1997).

The local MS marker density in the RYR1

gene region has now been increased from one

marker per 2.3 cm to one marker per 1.3 cm. The

largest gap in this region is now 5.3 cm

compared to the 8.6 cm and 7.3 cm in previously

reported maps (Ambady et al. 1997; Grimm

et al. 1997). This local high density genetic

map will facilitate ongoing QTL studies in this

laboratory focusing on this region of chromo-

some 6. The map will also facilitate the

construction of physical contigs to help identify

genes in this region of chromosome 6.

Acknowledgements

This research was supported by USDA grant 95±

37205±2314.

References

Alexander L.J., Rohrer G.A., Stone R.T. & Beattie C.W.

(1995). Porcine SINE-associated microsatellite mar-

kers: evidence for new artiodactyl SINEs. Mamma-

lian Genome 6, 464±8.

Ambady S., Mendiola J.R., Louis C.F. et al. (1997).

Development and use of a microdissected swine

chromosome 6 DNA library. Cytogenetics and Cell

Genetics 76, 27±33.

Archibald A.L., Haley C.S., Brown J.F. et al. (1995).

The PiGMaP consortium linkage map of the pig (Sus

scrofa). Mammalian Genome 6, 157±75.

Clamp P.A., Feltes R., Shalhevet D., Beever J.E., Atac

E. & Schook L.B. (1993). Linkage relationship

between ALPL, ENO1, GPI, PGD and TGFb1 on

porcine chromosome 6. Genomics 17, 324±9.

Cox D.R., Burmeister M., Price E.R., Kim S. & Myers

R.M. (1990). Radiation hybrid mapping: a somatic

cell genetic method for constructing high-resolution

maps of mammalian chromosome. Science 250,

245±50.

Ellegren H., Chowdhary B.P., Johansson M. et al.

(1994). A primary linkage map of the porcine

genome reveals a low rate of genetic recombination.

Genetics 137, 1089±100.

Fuji J., Otsu K., Zorzato F. et al. (1991). Identification

of a mutation in porcine ryanodine receptor asso-

ciated with malignant hyperthermia. Science 253,

448±51.

Green P., Falls K. & Crooks S. (1990). Documentation

for CRIMAP, Version 2.4, Washington University

School of Medicine, St. Louis, MO.

Grimm D.G., Goldman T., Holley-Shanks R. et al.

(1997). Mapping of microsatellite markers devel-

oped from a flow-sorted swine chromosome 6

library. Mammalian Genome 8, 193±9.

Harbitz I., Chowdhary B., Thomsen P.D. et al. (1990).

Assignment of the porcine calcium release channel

gene, a candidate for the malignant hyperthermia

locus to the 6q11->21 segment of chromosome 6.

Genomics 8, 243±8.

Lincoln S.E., Daly M.J. & Lander E.S. (1991). PRIMER:

A computer program for automatically selecting

PCR primers. MIT Center for Genome Research and

Whitehead Institute for Biomedical Research, Cam-

bridge.

MacLennan D.H. & Phillips M.S. (1992). Malignant

hyperthermia. Science 256, 789±94.

Paszek A.A., Flickinger G.H., Fontanesi L. et al.

(1998). Livestock variation of linked microsatellite

markers in diverse swine breeds. Animal Biotech-

nology 9, 55±66.

Paszek A.A., Schook L.B., Louis C.F. et al. (1995). First

international workshop on porcine chromosome 6.

Animal Genetics 26, 377±401.

Ponce de LeoÂn F.A., Ambady S., Hawkins G.A. et al.

(1996). Development of a bovine X linkage group

and painting probes to assess cattle, sheep and goat

X-chromosome segment homologies. Proceedings of

the National Academy of Sciences of the USA 93,

3450±4.

Rempel W.E., Lu M.Y., Mickelson J.R. & Louis C.F.

(1995). The effect of skeletal muscle ryanodine

receptor genotype on pig performance and carcass

quality traits. Animal Science 60, 249±57.

Rohrer G.A., Alexander L.J., Hu Z., Smith T.P.L.,

Keele J.W. & Beattie C.W. (1996). A comprehensive

map of the porcine genome. Genome Research 6,

371±91.

Rohrer G.A., Alexander L.J., Keele J.W., Smith T.P. &

Beattie C.W. (1994). A microsatellite linkage map of

the porcine genome. Genetics 136, 231±45.

Slonim D., Kruglyak L., Stein L. & Lander E. (1997).

Building human genome maps with radiation

hybrids. Journal of Computational Biology 4, 487±

504.

Sonstegard T.S., Ponce de LeoÂn F.A., Beattie C.W. &

Kappes S.M. (1997). A chromosome-specific micro-

dissected library increases marker density on

bovine chromosome 1. Genome Research 7, 76±80.

Weber J.L. (1990). Informativeness of human (dC-

dA) n. (dG-dT) n polymorphisms. Genomics 7, 524±

30.

255

Microsatellites from

microdissected swine

chromosome 6

ã 1999 International Society for Animal Genetics, Animal Genetics 30, 251±255