Allan M. Crawford," Grant W. Montgomery,* Craig A. Pierson,* Tim Brown,? Ken G. Dodds,? ... size (PIPER and BINDON 1982; DAVIS et al. 1982). The search for ...
Copyright 0 1994 by the Genetics Society of America
Sheep Linkage Mapping: Nineteen Linkage Groups Derived From the Analysis of Paternal Half-sib Families Allan M. Crawford," Grant W. Montgomery,* Craig A. Pierson,* Tim Brown,? Ken G. Dodds,? Sara L. F. Sunden,$ HannahM. Henry,* AndreaJ. Ede,* Peter A. Swarbrick," Thomas Berryman," Joanne M. Penty* and Diana F. Hill* *AgResearch Molecular Biology Unit, Department of Biochemistry and Centre f o r Gene Research, University of Otago, Dunedin, New Zealand, tAgResearch, Invermay Agricultural Centre, Mosgiel, New Zealand, fU.S. Department of Agriculture, Agricultural Research Seruice, Roman L. Hruska Meat Animal Research Center, Clay Center, Nebraska 68933
Manuscript received October 1, 1993 Accepted for publication March 2, 1994 ABSTRACT Nineteen linkage groups containinga total of 52 markers have been identified in the sheep genome after typing large paternal half-sib families. The linkage groups range in size2 markers from showingno recombination to a group containing 6 markers covering approximately 30 cM of the sheep genome. Thirteen of the groupshave been assignedto a sheep chromosome. Three groups contain markers from bovine syntenicgroups U2, U7 and U29, and one other group contains a marker thathas been mapped only in humans. The remaining three groups are unassigned. This information will provide a useful foundation for a genetic linkage map of sheep. "
G
ENETIC linkage maps are an important tool to identify genesassociated with production traits in large domesticanimals. Selection programs in farmanimals based on phenotypic measurementhave improved performance,butthegeneticalterationsunderlying most observed changes are unknown. An understanding of the structure and function of the genomesof domestic animals provided by genetic maps offers the opportunity to mapgenetic variation responsible for performance differences. Molecular markers linked to heritable traits will allowmore rapidselection of elite animals and eventually allow identification of those genes which influence phenotypic expression. One objective of the New Zealand SheepMap program is to develop a primary genetic linkage map for sheep for the purpose of mappinggenesinfluencingproduction.Production characteristics of particular interest includeprolificacy, wool production, disease resistance and growth. To evaluate strategies and approaches to marker assisted selection in sheep, we elected to study sheep carrying a single gene that increases litter size. Sheep derived from theBooroola Merino straincarry a mutation that segregates as a single autosomal dominant trait. The gene was named FecB by the Committee on theGenetic Nomenclature of Sheep andGoats (COGNOSAG 1989). It is additive for ovulation rate (+e number of ova shed at eachovulatory cycle) and partially dominant forlitter size (PIPERand BINDON 1982; DAVISet al. 1982). The search for markers linked to FecB combined the use of restriction fragment length polymorphisms for et al. 1992) and known candidate genes (MONTGOMERY the development of randomsheep microsatellites Genetics 137: 573-579 (June, 1994)
(CRAWFORD et al. 1991b).Markers were analyzed in large paternal half-sib families in which the Booroola gene was segregating. During the course of the exclusion study, 150 markers were screened in the pedigrees. Progress in the exclusion study was monitored by analyzing linkage between the restriction fragment length polymorphism (RFLP) and microsatellite markers. The genetic linkage map for sheepis rudimentary with only 10 markers assigned to four linkage groups (LEVEZIEL et al. 1991; PENTY andMONTGOMERY 1991; CRAWFORD et al. 1992) In this study we report the analysis of the markers in the Booroola pedigrees and the identification of 19 new linkage groups for sheep. MATERIALS AND METHODS All sheep were kept outdoors on pasture. The 12 families typed hadthe same structure in that a ram wasmated toa large number of ewes. DNA was isolated and the genotype analysis was undertaken using the ram and his daughters (range = 24-48 daughters).Each marker was first tested in the sireof he was heterozygous the familiesto determine whether or not a heterozygous at thetest locus. Only those families which had we could sire were then genotyped at that locus. To ensure that detect the most linkages possible for the least genotyping effort the12 families were given anorder of priority. Unless the marker was particularly uninformative a minimum of three familieswere genotyped. The three families chosen were those three with the highest priority that had a heterozygous sire. The families withthe top three priorities were genotyped with 38,31and 32 of the markers, respectively, whereas the families with the lowest priorities were genotyped with8 , 3 and 3 markers, respectively. A list of the RFLP and microsatellite markers used in the analysis is provided in Tables 1 and 2, respectively. A single protein polymorphismwas also used, transferrin,as previously et al. 1992). The method of RFLP analysis is described (TATE
574
A. M. Crawford et al. TABLE 1 Description of FWLP markers in ovine linkage groups Name
Abbreviation
TaqIisomerase Glucose phosphate Corticotrophin-releasing hormone Fibroblast growth factor basic XbaI Inhibin-PA TaqI Secreted phosphoprotein 1 RPl 1 (random sheep genomic DNA) RP29 (random sheep genomic DNA) Follicle-stimulating hormone-D
GPI CRH FGFB INHBA SPPl
Reference
Enzyme
J. PENTY (personal communication) SEEand MONTGOMERY (1992) (1993) PENTY and MONTGOMERY et al. (1992) HIENDLEDER MONTGOMERY et al. (1993) S. GALLOWAY (personal communication) S. GALLOWAY (personal communication) MONTGOMERY et al. (1990)
PVUII
TaqI TaqI EcoRI MspI
FSHB
TABLE 2 Description of microsatellite markers in ovine linkage groups
Name
Primers (5'
+
3')
FSHB~
TGGGATATAGACTTAGTGGC CAGTTTCTAAGGCTACATGGT HBBCHI~ CCTTACATCCAAAGGATGATTGGAG CCGTGGACAGAGGAGCTGGGTGGTC CCTGACTATAATGTACAGATCCCTC O M b GCAGAATGACTAGGAAGGATGGCA OLA-DRBp CTGCCAATGCAGAGACACAAGA GTCTGTCTCCTGTCTTGTCATC CTCCTCACACGGCTGCTGGG RHOb GTTAACTACAGTCTGTCCAGCTC CTATGATCACCTTCTATGCTTCC RBP3b CCCTAAATACTACCATCTAGAAG ACACTTGGAATTCAATAAATATGTGTTGG KAp8 GGACGCAACTGAGGACGCAACTG TAGGTCAGGAACCAGAGGCAGAG MAF4 TCCACGGGGTCTCAAAGAGTCG GGCTATAGTCCATGGAGTCGCAG MAF23 GTGGAGGAATCTTGACTTGTGATAG MAF36 CATATACCTGGGAGGAATGCATTACG TTGCAAAAGTTGGACACAATTGAGC GTAGACTACTCATGAAAATCAGGTCTTAGG MAF50 GGGACATGCAGCTATACACTTGAG CTCATGGAATCAGACAAAAGGTAGG MAF64 AATAGACCATTCAGAGAAACGTTGAC CACGGAGTCACAAAGAGTCAGACC MAF70 GCAGGACTCTACGGGGCCTTTGC TAGAATGTCATGTTCTCAGCATTCCC MAF92 AACCCATGAATCATCTCTAACTACCTC TCATGCACTTAAGTATGTAGGATGCTG MAF209 GATCACAAAAAGTTGGATACAACCGTGG AATGCAGGAGATCTGAGGCAGGGACG MAF214 GGGTGATCTTAGGGAGGTTTTGGAGG OarFCB48 GACTCTAGAGGATCGCAAAGAACCAG GAGTTAGTACAAGGATGACAAGAGGCAC OarFCB266 GGCTTTTCCACTACGAAATGTATCCTCAC CACCACATACCAAACACACAGCCTGC OarFCB304 CCCTAGGAGCTTTCAATAAAGAATCGG CGCTGCTGTCAACTGGGTCAGGG TACTAAAGAAACATGAAGCTCCCAC OarAE54 GGAAACATTTATTCTTATTCCTCAGTG TGCAAGAAGGGCAGACCTTGGAG OarAE64 CAGACCACTCTCTTCCCTCCACG OarAElOl TTCTTATAGATGCACTCAAGCTAGG TAAGAAATATATTTGAAAAAACTGTATCTCCC AATTGCATTCAGTATCTTTAAACATCTGGC OarHH35
No. of Annealing Accession alleles PIC" temp number
Source
OarHH55 OarHH56
TCCACAGGCTTAAATCTATATAGCAA GAGCGGTGTAGTAGAAAATAGAAATCGACC GTTATTCCATATTCTTTCCTCCATCATAAG CCACACAGAGCAACTAAAACCCAG GCAACCCACTCATCTCTCCGTGTC GAAAACTTAAGTTCCAGCTATTAAAATAGC
MOOREet al. (1992)
2
0.36
63
3
0.46
60
6
0.78
58
X12672
Bovine
MOOREet al. (1992)
11
0.74
62
M33306
Ovine
BLATTMAN and BEH (1992)
5
0.61
60
M2 1606
Bovine
MOOREet al. (1992)
4
0.61
55
M20748
Bovine
MOOREet al. (1992)
11
0.85
50
X05639
Ovine
WOODet al. (1992)
19
0.90
65
M61729
Ovine
BUCHANAN et al. (1991)
4
0.52
65
M38719
Ovine
SWARBRICK et al. (1990)
13
0.85
65
M61728
Ovine
et al. (l99lb) SWARBRICK
6
0.41
61
M77377
Ovine
SWARBRICK et al. (1992)
11
0.80
64
M62993
Ovine
et al. (1991a) SWARBRICK
17
0.90
64
M77199
Ovine
BUCHANAN CRAWFORD and (1992a)
6
0.62
65
M61730
Ovine
et al. (1991a) CRAWFORD
8
0.79
63
M80358
Ovine
(1992b) BUCHANAN CRAWFORD and
6
0.60
60
M88160
Ovine
BUCHANAN CRAWFORD and (19924
11
0.76
60
M82875
Ovine
BUCHANAN et al. (1993)
7
0.57
63
LO1534
Ovine
BUCHANAN CRAWFORD and (1993)
9
0.54
63
LO1535
Ovine
BUCHANAN and CRAWFORD (1993)
8
0.82
63
L11048
Ovine
et al. (1993) PENTY
15
0.86
63
L13869
Ovine
EDEet al. (1993)
9
0.72
63
L13692
Ovine
MONTGOMERY et al. (1993)
7
0.70
63
L12554
Ovine
et al. (1993) HENRY
11
0.80
63
L12555
Ovine
HENRY et al. (1993)
12
0.73
63
L13693
Ovine
MONTGOMERY et al. (1993)
5
0.36
61
L13871
Ovine
EDE et al. (1993)
M20185
Bovine
Caprine WOODet al. (1993)
ATGAAAATATAAAGAGAATGAACCACACACGG
OarHH41
Reference
575
Sheep Linkage Map TABLE 2 Continued ~~~~~
No. Annealing Accession of number Source temp PIC' alleles
Name
Primers (5' + 3')
OarHH62
TAATGAGTCAAACACTACTGAGAGAC AATATATAAAGAGAAAAGCTGGGGTGCC CTCTAGAGGATCTTGTAATAATCTATAAAGG GTTAATTTACAGTGTTGTGTTAGTTTCAGC CGCAGTATTTTAGTCCTTTTAATAATGGC TCGTAAGAGTGGACACAACTGAGCG GGCCTCTCAAGGGGCAAGAGCAGG CTCTAGAGGATCTGGAATGCAAAGCTC ATGAACTAACCTCCGTAGACCCAGC CCGGAAGTCCATAAGAGCTATTCTTAACC CTCTAGAGGATCACAGAGAGTCGG GCAGAAACATTTTTTTCCTTCAATATAGTTTCCC AGTGTGACTAGAGAACTAAATTTTGAAGGTC TATTTTTCCATCAAAAAGAACTCTATAGGGC CACAAAGAGTCAAGACATGCCCGAG ATTGGCAGACATCCTTTCACTCCC ACTCTTTACTACTGTACCACCTAGG ATATTGAGGGCTTAAGTTATGTCTTC GTATGTATTTTTCCCACCCTGC GAGTCAGACATGACTGAGCCTG GAGCAAGGTGTTTTTCCAATC CATTCTCCAACTGCTTCCTTG TTGAGCACAGACACAGACTGG ACTGAATGCCTCCTTTGTGC TCTATACACATGTGCTGTGC CTTAGGGGTGAAGTGACACG CTTAAAATCTGTCTTTCTTCC TAGTGTGTATTAGGTTTCTCC GGAAGCAATGAAATCTATAGCC TGTTCTGTGAGTTTGTAAGC GCTTGCTACATGGAAAGTGC CTAAAATGCAGAGCCCTACC CTTGATCCTTATAGAACTGG ACACAAAATCAGATCAGTGG GGAAATACCTTATCTTTCATTCTTGACTGTGG CCTTCTTTCTCATTGCTAACTTATATTAAATATCC
OarVH4 OarVH34 OarVH72 OarVH98 OarVHllO OarVH116 OarVH117 OarVH 130 BM1258 BM1824 BM6438 ILSTSOO2 ILSTS004 ILSTS005 ILSTSOll ILSTSOl3 JP15
Reference
11
0.80
63
L13872
Ovine
EDEet al. (1993)
7
0.70
58
L13873
Ovine
EDEet al. (1993)
9
0.68
63
L12559
Ovine
et al. (1993) PIERSON
7
0.76
63
L12548
Ovine
PIERSON et al. (1993)
9
0.74
50
L12549
Ovine
HANRAHAN et al. (1993)
10
0.80
58
L12550
Ovine
HANRAHAN et al. (1993)
9
0.63
63
L12551
Ovine
HANRAHAN et al. (1993)
11
0.75
63
L12552
Ovine
HANRAHAN et al. (1993)
8
0.80
55
L12553
Ovine
et al. (1993) HANRAHAN
4
0.48
63
Bovine
C. BEATTIE
5
0.68
63
Bovine
6
0.77
63
Bovine
5
0.72
50
Bovine
(personal communication) C. BEATTIE (personal communication) C. BEATTIE (personal communication) KEMP et al. (1992)
6
0.72
50
Bovine
KEMP et al.
6
0.51
50
Bovine
BREZINSKY et al. (1993a)
5
0.66
50
Bovine
BREZINSKY et al. (1993b)
5
0.70
50
Bovine
BREZINSKY et al. (1993b)
6
0.68
63
Cervine J. PEMBERTON (personal communication)
(1993)
'PIC = polymorphic information
content. hormone-a; HBB-CHI, The firstseven microsatellite markers are associated withknown genes which are as follows: FSHB, follicle-stimulating goat hemoglobin-a; OCAM, opioid-binding cell adhesionmolecule;OLA-DRBp,ovine DRB pseudogene; RHO, bovineopsin gene; RBP3, retinol-binding protein3, interstitial; KAF'8, glycine and tyrosine rich keratin gene.
et al. (1992). The probesused fully described by MONTGOMERY for RFLP detection were as follows: corticotrophin-releasing hormone (CRH),ovine cDNA probe, pCRF3l supplied by K. IMOTO(FURUTANI et al. 1983); follicle-stimulating hormone-P (FSHB), bovine cDNA clone supplied by R. MAURER (MAURER and BECK1986); inhibin-PA (INHBA), ovine cDNA clone, pFLBA-3, supplied by D. TISDALL(personal communication); secreted phosphoprotein 1 SSPl, bovine cDNA clone, OP-12, supplied by L. W. FISHER (KERRet al. 1991);glucose phosphate isomerase (GPI), porcine cDNA clone, pGPI8R, supplied by I. E~ARBITz (DAVIES et al. 1988); fibroblast growth factor, basic (FGFB), bovine cDNA clone pJJ11.1 supplied by J. A. ABRAHAM (ABRAHAM et al. 1986); RPl1 and RP29, are anonymous ovine personal communication). genomic DNA clones (S. GALLOWAY, Microsatellite alleles were amplified using the polymerase chain reaction (PCR). The reaction took place in a total volume of 10 pl and contained thefollowing constituents at the final concentrations indicated in parentheses: dNTP (200 VM), TrisHC1, pH 8.8 (45 mM), (NHJ2 SO, (11 mM), MgCl, (4.5 mM), 2'-mercaptoethanol (6.7 mM),EDTA (4.5 p ~ ) , spermidine (0.25 mM), bovine serum albumin (200 pg/ml), unlabeledprimer (400 nM), labeled primer(20 nM), and Taq DNA polymerase (0.5 unit/reaction). The DNA to be amplified was at an approximate final concentration of 10
ng/pl.One of theprimers was end-labeled using either [y'P]ATP or [y3P]ATP and T4polynucleotide kinase (New England Biolabs). Thermal cycling conditions for the PCR were as follows: seven cycles of denaturation at 95", 30 sec and annealing temperature as in Table 2, 1 min followed by 20 cycles of denaturation at go", 30 sec and annealing temperature as in Table 2, 1 min, using either a Perkin-Elmer Cetus or a Techne PHC3 (Cambridge,United Kingdom) thermal cycler. No extension step was used. PCR products were analyzed on denaturingsequencing gels and thebands of DNA visualized by autoradiography. The sire was always assigned the AB genotype and the daughterswere assigned as either AA, AB, AC, BB or BC where the C allele was defined as any allele other than those of the sire. This simplified genotype scoring as it meant that each allele of the multiallelic microsatellites did nothave to beidentified and all sizing was related to the sire's genotypewhich was included on all gels containing members of his family. The allele passed to a particular daughter by its sire was determined (A or B).Where this was not possible the daughter was not included in the linkage analysis. For each family the remaining daughterswere then assigned to one of two classes. One of these contained those daughters that hadreceived an A allele from their sire at both loci or that had received a B
576
A. M. Crawford et al. TABLE 3
TABLE 4
Linkage groups containing three or more markers in the sheep genome presented as matrices with recombination fraction in the upper triangle and lod score in the lower triangle
Pairs of markers showing genetic linkage in the sheep genome
Linkage group A
OarFCB48
OarFCB48 OarVH4 OarVH116 OarVH98 MAF209 FGFB 2.1
9.2 2.5 4.6 6.0 2.2
Linkage group B OarVH72 OarAE54 OarFCB266 OarVHll7 RBP3 Linkage group C
OarVH4
OarVH116
OarVH98
0.10
0.19 0.00
0.20 0.05 0.14
8.4 8.3 7.3
OarVH72
2.5 5.2 1.9
OarAE54
NCS
4.2
4.2 2.1 0.8
3.9
BM6438
0.18 0.09 0.07 0.29
J
K L M
0.16 0.11 NCS~
N 0 P
0.17 0.05
3.4
OarFCB266
OarVH117
NCS
0.20
0.00
NCS
0.20 0.5
NCS
I
MAF209 FGFB
NCS
0.18
Q R S
RBP3 0.28 0.15 0.00 NCS
NCS
KAP8
MAF64
ILSTS004
0.03
0.14 0.17
0.30 0.25 0.23
7.1 5.4 1.3
Linkage group D
FecB
FecB OarAElOl SPPl OarHH55
17.3 1.4 9.3
1.0 1.4
3.4
OarAElOl 0.13
SPP 1
OarHH55
0.14 0.00
0.20 0.05
3.6 15.4
NCS NCS
Linkage group E
MAF4
MAF4 TF BM1824
9.9 6.5
6.9
Linkage group F
MAF23
OarVHl30
OarVH34
MAF23 OarVH130 OarVH34
0.05 4.2 9.4
0.07 0.06
Linkage group G
TF
BM1824
0.00
0.03 0.00
8.7
BM1258 OLA-DRBp
OLA-DRBp BM1258 OarHH56
0.11 3.6 5.3
Linkage group H
JP15
JP15 OCAM OarVHllO
4.3 3.1
OarHH56 0.00 NCS NCS
OCAM
OarVHllO
0.16
0.20 0.20
3.2
a NCS = the two markers were not used in a n y common families so genetic linkage could not be tested.
allele from their sire at both loci, while the other class contained all other daughters whose sire’s alleles at bothloci could be determined.
Markers
ILSTSOll, CRH OarFCB304, RHO OarHH35, INHBA MAF92, MAF36 MAF70 MAF50, ILSTSOOP, GPI OarHH62, MAF214 ILSTS005, OarAE64 OarHH41, RPll HBBCHI, FSHB ILSTSO13, W2B
fraction
LOD score
0.16 0.12 0.11 0.10 0.08 0.08 0.04 0.00 0.00 0.00 0.00
4.0 4.8 4.5 4.1 8.8 4.1 4.8 21.4 5.4 4.5 3.3
Linkage was carried out in the same manner as for the phase-unknown double backcross ( O n 1991; DODDSet al. 1993). For this type of data the lod score (MORTON1955) is calculated using the formula
=
BM6438 KAP8 MAF64 ILSTSOO4
Recombination
group
1.3
5.2
Linkage
x [(n
- l)log,,(z)
+ iog,,(ek(i - e).-
k+
en- k(l - e)”]
/omder
where 8 is the recombination fraction, n is the size of a particular family and k is the number of daughters in the family who received either an A at both loci or a B at both loci from their sire. The lod score was calculated for 0 at I c M intervals to find the maximum lod score, and corresponding recombination fraction estimate. This method allows the linkage analysis to be carried out without requiring estimates of allele frequencies, and is identical to the analysis performed by CRIMAP (LANDER and GREEN1987) for data with this typeof pedigree structure. A maximum lod score of at least 3 is taken as evidence of linkage in human studies ( O n 1991), and this critical value is also appropriate for the sheep genome which is of similar size to that of the human. RESULTS
Using two point linkage analysisall markers that shared a commonheterozygous sire weretested for significant linkage. A total of 19 linkage groups were identified and are presentedin Tables 3 and 4.For two markers to be accepted as significantlylinked we required a minimum lod score of 3.0 and that the markers were segregating in at least two half-sib families. There was one exception to this ruleincluded in the linkage groups. The inclusion ofBM1258 in the OLA-DRBp, OarHH56 linkage group was based on data from only one family as only one heterozygous sire was found in common between the markers. Those markers linked to another marker or group of markers by a low lod score (
