Open Access

A physical map of a BAC clone contig covering the entire autosome insertion between ovine MHC Class IIa and IIb

Contributed equally
BMC Genomics201213:398

DOI: 10.1186/1471-2164-13-398

Received: 21 January 2012

Accepted: 3 August 2012

Published: 16 August 2012

Abstract

Background

The ovine Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of the host to infectious diseases. Compared to human and mouse, the ovine MHC is interrupted by a large piece of autosome insertion via a hypothetical chromosome inversion that constitutes ~25% of ovine chromosome 20. The evolutionary consequence of such an inversion and an insertion (inversion/insertion) in relation to MHC function remains unknown. We previously constructed a BAC clone physical map for the ovine MHC exclusive of the insertion region. Here we report the construction of a high-density physical map covering the autosome insertion in order to address the question of what the inversion/insertion had to do with ruminants during the MHC evolution.

Results

A total of 119 pairs of comparative bovine oligo primers were utilized to screen an ovine BAC library for positive clones and the orders and overlapping relationships of the identified clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 368 positive BAC clones were identified and 108 of the effective clones were ordered into an overlapping BAC contig to cover the consensus region between ovine MHC class IIa and IIb. Therefore, a continuous physical map covering the entire ovine autosome inversion/insertion region was successfully constructed. The map confirmed the bovine sequence assembly for the same homologous region. The DNA sequences of 185 BAC-ends have been deposited into NCBI database with the access numbers HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826.

Conclusions

We have constructed a high-density BAC clone physical map for the ovine autosome inversion/insertion between the MHC class IIa and IIb. The entire ovine MHC region is now fully covered by a continuous BAC clone contig. The physical map we generated will facilitate MHC functional studies in the ovine, as well as the comparative MHC evolution in ruminants.

Keywords

Ovine MHC OLA Physical map BAC Comparative mapping

Background

The mammalian Major Histocompatibility Complex (MHC) harbors genes involved in overall resistance/susceptibility of animals to infectious pathogens, including viral, bacterial, internal and external parasites. Pathogens serve as sources of selection pressure to their host animals, and the hosts are forced to develop effective strategies to fight against the pathogens in various environments. Such co-evolutionary struggles may have left distinct marks in the genome of each species involved, and mammalian MHC regions have been shaped into clusters of immunological gene families by such host-pathogen interactions, probably via functional gene duplications [13]. The implications of ovine MHC molecules in providing protection against pathogens [48] and the associated structures of the artiodactyl’s MHC region in general have led to a number of studies into the sheep MHC [915].

The ovine MHC, also called ovine leukocyte antigen (OLA), is located on the long arm of ovine chromosome 20 (OAR 20q15–20q23) with a similar structure and organization to that of human and other mammals [16]. The literature shows that MHC genes play vital roles in resistance of animals to foot rot [17], parasites [9], and bovine leukemia virus [7]. To date, the majority of studies on the structure and organization of the ovine MHC have focused on the gene content and polymorphism of the class II region [1823]. Although most loci in the sheep MHC are found to be homologous to their counterparts in the human MHC [12, 21, 24, 25], there are significant differences. Examples of such differences include the DP loci in human being replaced by DY in sheep [19, 21, 26, 27], and the number of DQA loci varying significantly among sheep breeds [20, 22, 28].

Compared to human and mouse, the structure of the sheep MHC is interrupted by a piece of ~14 Mb autosome insertion, possibly via a hypothetical chromosome inversion (inversion/insertion) in the class II region, similar to that of cattle [24, 2932]. The inversion/insertion constitutes ~25% of ovine chromosome 20, which spliced the MHC class II region into IIa and IIb. The significance of such an insertion in relation to the ovine MHC functions remains unknown. The evolutionary consequence of such an event is also worthy of attention, because some of the ovine-specific MHC loci like DY, and Dsb are located near the boundary region of the inversion/insertion. We previously constructed a physical map of BAC clone contigs covering the ovine MHC except the autosome insertion region [12, 13], and a high accuracy sequence map of sheep OLA was accordingly constructed [14].

With the initial release of sheep whole genome reference sequences by the International Sheep Genomic Consortium (ISGC), much more genome sequence information is now accessible for functional and comparative studies [33]. Nevertheless, the sequence map would serve the research community even better if it is cross-referenced/checked for accuracy in DNA sequence and assembly, at least for some chromosome regions, by an alternative approach. In this regard, the detailed information is still not fully available for the gene structure, organization, and DNA sequence for the ovine chromosome region between OLA class IIa and IIb [12, 14, 27].

In this paper, we describe the construction of a BAC physical map covering the entire autosome insertion between ovine MHC class IIa and IIb. Because ovine and bovine species share the consensus structure and organization in the entire MHC region [24, 2932], we used comparative approaches to screen a sheep BAC library with 119 bovine oligo nucleotide primers designed from the bovine genomic sequences for the consensus region. The order and overlapping relationship of the identified BAC clones were determined by DNA fingerprinting, BAC-end sequencing, and sequence-specific PCR. A total of 108 effective overlapping BAC clones were selected to fully cover the region between class IIa and IIb. The physical map we constructed will help to generate ovine MHC sequencing map with a high level of accuracy, which in turn will facilitate MHC functional and comparative MHC evolution studies in ruminants.

Methods

Comparative design of oligo primers

A BAC library was previously constructed using the genome DNA from a male Chinese merino sheep, with a total of 190,500 BAC clones and an average insert length of 133 kb [12, 13]. To screen the BAC library for positive clones in the target genome region between ovine MHC class IIa and IIb, we adapted a comparative strategy to design bovine oligo nucleotide primers using the bovine reference DNA sequences in the consensus genome region [34]. At the time this study was conducted, no sheep genomic sequence was publicly available for the genome region of our concern. Bovine DNA sequences of homologous genes, exon, intron, or partial STS sequences were acquired from the NCBI website (http://www.ncbi.nlm.nih.gov/genome/sts/). Primers were designed along the bovine MHC region between class IIa and IIb, approximately 80–160 kb apart between two neighbor loci using the software Prime Primer 5.0 (Biosoft International, CA). A total of 119 bovine primer pairs were designed for screening the sheep genomic BAC library (Table1).
Table 1

Comparative bovine primers used for identification of the positive ovine BAC clones in the genome region between MHC Class IIa and IIb *

Name

Gene symbol

Primer sequence (5’→3’)

Product(bp)

Bovine template sequence

Positive Ovine BAC clones

S001

VPS52

F: ATCAATCAGACGATTCCCAACG

246

UniSTS:279053

12 H14;12I12;12 J14; 12 K14;120P21

  

R: ATCAGAAACACAAGCTGCTCCT

   

S002

ZBTB22

F: TCCTACGACTTACTCCCTCC

250

UniSTS:66823

12I12;12 J14;258 F9; 289 G18

  

R: GGGTCAGGTGGTTGTAGTCT

   

S003

KIFC1

F: GAGACTGTCCGAGACCTGCT

1242

UniSTS:BV104878

170 G9;217 M14;289 G18

  

R: CTGTGACTACGCGACGAGC

   

S004

Loc100139397

F: GGTCATCATGGAGGCAGTCT

756

Exon 6: NC_007324

19 H17

  

R: CGTTCTCCTAAGCCATATGC

   

S005

BAK1

F: CATTGCATGGTGCTAACCGA

293

Exon 6: NC_007324

None

  

R: CAAGCTCAGCCTTCCAGAAC

   

S006

IHPK3

F: ATGTATGAGAGCTTGGCACG

1000

UniSTS:267905

212D3

  

R: TCAGCTTGTACTCTTCCAGGG

   

S007

LEMD2

F: ACGTCTACCGCAACAAGCTG

227

ENSBTAE00000168818: Exon 1

None

  

R: GTCTCCGATGTCACCGTAGG

   

S008

Loc790333

F: GACTGCGAGGTGCCGAAGAA

776

Exon

94 M24;114B22

  

R: GTGGACGGCTACACCTGCAA

   

S009

HMGA1

F: CTCATGCTCTCATTCGGACA

625

ENSBTAE00000364012: Exon 6

57 M5

  

R: CAGAACAGGAGGCAATGAGG

   

S010

NUDT3

F: TGAAGTGGAGAGCCTCACAA

688

ENSBTAE00000213256: Exon 5

14E10;300 G8

  

R: CTTCTCAGCAGACGATGGAC

   

S011

COX5B

F: GTCTCCGTGGTGCGCTCTAT

324

ENSBTAE00000098033: Exon 1,2

130 G21;130 M2;170 K16

  

R: GGTGTGGCACCAGCTTGTAA

   

S012

PACSIN1

F: AAGCCAGCAACAGTAGCAGC

683

ENSBTAE00000336066: Exon 10

253I24

  

R: TCGTTACCTGGAGACCAAGC

   

S013

C6orf106

F: AGTGAGCGGCTGAGAGAGTT

266

ENSBTAT00000048861: Exon 1

None

  

R: AACTCGGAGATGAGCACGTC

   

S014

SNRPC

F: CCAATGATGAGACCTCCTGC

147

ENSBTAT00000034155:Exon 6

119P19;157 K19;223 N7; 227 J17;232 G24

  

R: CAGAGTCACAGCACCATGAT

   

S015

TAF11

F: TGGATGTGTGTGAGAAGTGG

561

ENSBTAT00000022463: Exon 5

194 L19;215 J4;232 G24;234C5

  

R: TCATGGTGGAGTATCACAGG

   

S016

ANKS1A

F: CGAGGAATGGCCACAAAG

894

UniSTS:BV105378

124P23;320A1

  

R: ATCGGTCTTGCCAAACAAAG

   

S017

TCP11

F: ATCAGCGGATCCACTTGTTC

373

ENSBTAT00000022467: Exon 11

24D11

  

R: CTGGAGCTCACACACGAGGT

   

S018

DEF6

F: ACCACCAGCAGCTCCTTCAC

496

ENSBTAT00000036152: Exon 11

21 M13;66I6;124 K16; 193E6;206 L10

  

R: CCTGGCTTGCTTGTTGACTC

   

S019

PPARD

F: GTTCCATGGTCACCTTCTCC

353

ENSBTAT00000023319: Exon 8

28D20;152A4

  

R: CCGTGAATCTCGCTTCTCTT

   

S020

TEAD3

F: CCCATCACAGCTGGATTTTA

145

UniSTS:180986

None

  

R: AAATGAAGTACTGTGCCCCC

   

S021

Loc540812

F: TGCACTGCAACTTCCTGAAC

263

Exon

95D10;119O20;158O6

  

R: GCACTGCAGGCTGACTATGA

   

S022

SRPK1

F: CAGACACTTACAGGACGTGG

273

ENSBTAT00000022396: Exon 11

269D12;285I5

  

R: TGAAGACTGGCACATCATGG

   

S023

SLC26A8

F: ACATCAGCACCGTCAGTCACC

222

UniSTS:476830

26A21;121O15

  

R: AGGCGATAGAGGACAAACCACAC

   

S024

MAPK14

F: GAATGGATAACAAAACACTT

196

UniSTS:279403

26A21;121O15

  

R: AGGCGATAGAGGACAAACCACAC

   

S025

MAPK13

F: AGAAGCTCAATGACAAGGCG

606

UniSTS:269171

121O15;154 M16

  

R: TTCCATTCGTCCACTGTGAG

   

S026

BRPF3

F: GACGCCTGCATCGTATTAGC

575

ENSBTAT00000017711: Exon 1

154 M16;250 L24; 278B11;281D9;300 J5

  

R: AGCCAGGTTGCAGATGTCAC

   

S027

PNPLA1

F: TCCTGAACGCTGTCAACCGA

449

ENSBTAT00000055658: Exon 7

78 M7;153 F9;268E18; 319O4;337 K13

  

R: CAGGTGGCTGTGCAGGTGAT

   

S028

Loc790226

F: CCATGACTCCGTAGACAAGA

483

Exon

3O16;9 G2;9 G3;9 H8; 10 N2;15B13;26D1

  

R: ACTGCCATAGCTACTGCTGC

   

S029

KCTD20

F: CGATGCAATCACTAAGCTGG

834

ENSBTAT00000027439: Exon 8

None

  

R: GCAGTTCTCATCCTTCGCAC

   

S030

RPS4Y1

F: TGCCAGCCTCTTGTCTCTCT

430

ENSBTAT00000036142: Exon 2

2A3;11 H24;63 N7; 82 N20;97O2;120P24

  

R: TACACCTGAGGAGGCCAAGT

   

S031

CDKN1A

F: GGATCGCTAAGAGCCGGACA

861

ENSBTAT00000011001: Exon 3

None

  

R: GGCAGTCGCTGCTTGAGGTA

   

S032

PPIL1

F: AATGGTCAATGCGCCTGCTT

888

ENSBTAT00000003071: Exon 4

30O17;139 K9;198 M20;271C5

  

R: CACCAACGGCAGCCAGTTCT

   

S033

PI16

F: CCTAGCAACAGAAGCCTCAA

461

ENSBTAT00000002703: Exon 5

54O24

  

R: AGGCCAAGATCTCACTGCAA

   

S034

FGD2

F: CACCTTGGTGACCAACATTC

414

ENSBTAT00000018834: Exon 16

304 K7;318I17

  

R: ACTGCCATAGCTACTGCTGC

   

S035

PIM1

F: AAGCACGTGGAGAAGGACCG

490

UniSTS:463218

None

  

R: GACTGTGTCCTTGAGCAGCG

   

S036

TBC1D22B

F: CTGTCCACCACTCCATGTCT

539

ENSBTAT00000018938: Exon 13

5 K4;26A20;49B1;98 G9

  

R: GGACATTCGGACGTGTAACT

   

S037

RNF8

F: TCTGAATGGTGTCTGGCTGA

708

ENSBTAT00000010959: Exon 3

None

  

R: TTCTCGAGCTGCTCCACTCT

   

S038

Loc509620

F: AGTGGCACACCGAAGCTC

666

UniSTS:267349

25P1;103D16;207 L11; 271 M7

  

R: AACTTCCTCTTGAAGCTTTTGC

   

S039

C23H6orf129

F: GGCAAGAGAACCGCAAGAAC

281

ENSBTAT00000016009: Exon 4

25P1;103D16

  

R: GCACGAAGTCCTTCTGGAGC

   

S040

MDGA1

F: TCTTGGCGTTGCAGAGATGA

228

ENSBTAT00000047505: Exon 16

None

  

R: TGTGCGTGTGTCGAACAACC

   

S041

ZFAND3

F: CGATTGGTTTAATTTTTTTTTTCA

200

UniSTS:34520

159 K21;185 L24;235B3

  

R: TGTGAAGTTTGTTAAATGTAAGGAA

   

S042

BTBD9

F: GATAGGTCTTACGCTGTTAG

155

UniSTS:279369

None

  

R: GAATGTACAGAATAGAAGTG

   

S043

Loc781915

F: AACCTCAAGTGCCTCTCCAG

714

Exon

67D11;70 N21;76E1; 240 K15;240O16

  

R: AACAAGTGTAGCCAGCCATC

   

S044

GL01

F: GATAGGTCTTACGCTGTTAG

155

UniSTS:279369

None

  

R: GAATGTACAGAATAGAAGTG

   

S045

Loc525414

F: GAAGAAGAGGTGATCGGTGTAGAG

216

UniSTS:476833

8 J2;13E21;24 K16; 24 N15;28 L5;112 N3

  

R: TTTCTCCTTCCCATACATTTCTGTG

   

S045b

GLP1R

F: CGAGTGTGAGGATTCCAAGC

418

Exon 4, 5 and intron

80 G15;138P3

  

R: GTAGCCCACCGTGTAGATGA

   

S046

C23H6orf64

F: GTCACAGCCACCATGGAGTC

415

ENSBTAT00000001425: Exon 2

19 F4;80 G15;138P3; 156B12; 336 L24

  

R: CGCAAGCTGTTCTCAGTCAA

   

S047

KCNK5

F: CTCCGACTCTGTGCTGGTGA

774

ENSBTAT00000014756: Exon 5

None

  

R: TACCACGCCTTGTACCGCTA

   

S048

KCNK17

F: AGAGTCCAGGCTCCTTCTAT

493

ENSBTAT00000013646: Exon 5

None

  

R: CTGCTATCCTCAGAGTTCCA

   

S049

Loc100139627

F: GTGGAGGGAACCTGCGGCAC

344

NC_007324.3: designed online

3 L3;51O8;189 L22; 253I5; 270 L14

  

R: AGGCCTCGGAAGAGCCCTGG

   

S050

Loc100138924

F: CTTGGTCTTGCGGGCCCCTG

493

NC_007324.3: designed online

145 G9;146 H11

  

R: CCAGGCTCTAGCCCTGCCCA

   

S051

DAAM2

F: CAGGGAGTGCTCTCAAAGGTAAAGG

307

UniSTS:476834

None

  

R: TCCTCCAGCCTGACTTCTCCTTC

   

S052

MOCS1

F: GGTCCAGGAAGGCTGAAGTG

661

ENSBTAT00000013792: Exon 11

None

  

R: GAAGGACGGATGGCTATGGT

   

S053

LRFN2

F: TTGTCATACACGGCGGTCCT

493

ENSBTAT00000023907: Exon 1

77E2;220 J8;325 J12; 325 J13

  

R: AGCTGAGCCTCGACCACAAC

   

S054

UNC5CL

F: TGACCAACGAGCAGCCACAC

278

UniSTS:476835

None

  

R: GCAGCAGGAGGAGCCAGAAG

   

S055

NFYA

F: GCCGATGAAGAAGCTATGAC

550

ENSBTAT00000013080: Exon 10

76 K24;118P22;136B19

  

R: CATGAGATGGAGCTTCCTTG

   

S056

TREM2

F: ACAACTCCTTGAAGCACTGG

229

ENSBTAT00000009568: Exon 2

86A4;178 L4;208 M19; 282 F4

  

R: TGGAGGCTCTGGCACTGGTA

   

S057

TREM1

F: CATCATTCCTGCAGCATGTG

515

ENSBTAT00000023397: Exon 4

30C8;73 K17;75A11; 75I21

  

R: GGCTGTGCCAGGTCTTAGTT

   

S058

LOC783024

F: CTGAGGACCAAGGCCATGCT

216

Exon

None

  

R: TGGTGTGGCACTGCAGGAAG

   

S059

FOXP4

F: AATTATCGCTCCAAGAGATTCCAC

250

UniSTS:384935

112I1;144 K17;181 F9; 299P14;314 F18

  

R: CCCATCCTTGTCTCCTCTTTACAT

   

S060

MDFI

F: GCTGTGTCCACTGCATCTTG

256

ENSBTAT00000025763: Exon 4

70B14;166C6;181 J11; 202B12; 229A10

  

R: GGTCAGGAGGAGAAGCAGAG

   

S061

PGC

F: GAAATTCTCTGCTAAACCCCTTCA

268

UniSTS:385581

14 G18;24O7;24O10; 103 G9; 139 N14

  

R: TCATCTAAGCAGAAACACCAGTAAATG

   

S062

USP49

F: GATGGAGTTCATGTAGCAGGTGTT

260

UniSTS:385828

None

  

R: GGAGCGCAAGAAGGAGGAG

   

S063

BYSL

F: TCAGAGGACCTGGAAGTGGA

538

ENSBTAT00000013326: Exon 7

3 M12;98 J10;182 F10

  

R: CTCTCATGCACAGCAGTGGA

   

S064

TAF8

F: TGGAGGAAGGAACTTGGTCACAGAG

228

UniSTS:476836

103 M11;133 J10;146 L22

  

R: GGTGCTTGAGGTTCGTTGAGTTGAG

   

S065

MGC137036

F: GAAGCAGGACCGTGAGCAGA

238

ENSBTAT00000017035: Exon 2

100O15;117E7;133 J9; 146 L22;171 L22;176P6

  

R: CTACGAGCGCCACAAGACCA

   

S066

TRERF1

F: GTGTGTCTGTTGCTGCGGTG

643

ENSBTAT00000020376: Exon 1

1O22;17 J12;79 H15; 81 J21;100O15;259 L15

  

R: TGGTCTAGGCTTGGCTGTTG

   

S067

Loc786000

F: TGGCAAGATGGCGGTGCCAG

379

NC_007324.3: designed online

6P21;32P14;142C8; 162E5;195C23;227D22

  

R: AGCAGCCTTGGCCCCACTCT

   

S068

UBR2

F: CTGCAAGCAACTGACCTCAC

169

ENSBTAT00000007833: Exon 2

6P21;129B6;162E5; 163E23;177 M6

  

R: CCAACTCAGGATCTTCACCA

   

S069

PRPH2

F: GTAGTGGACTCCAGGAACTTCG

232

UniSTS:279013

26 J6;26 L8;29 M14; 127A7;134B12;177A2

  

R: ACCACAGAGTCACCTGCTGAGA

   

S070

Loc540169

F: ATGAAAGGGTCAGGCGAAC

130

UniSTS:94727

144A13;164 L3;164 M2;164 M3;172O18;185 N10

  

R: ACAGAGCCGCTAACCGTG

   

S071

CNPY3

F: GAACAGTGGTCTGGCAAGAA

214

ENSBTAT00000021132: Exon 10

98 J16;172O18;185 N10;189O8;289 J21

  

R: GTTAGGCTCAGAGCTCGTCA

   

S072

CUL7

F: TTTCGACCTCGCTCTGAGTT

1,000

UniSTS:270008

74C2;189O8;289 J21; 325 K12

  

R: CTCCAGCATGTGCCAGTG

   

S073

PTK7

F: GACTCAGGAGCCTTCCAGTG

531

UniSTS:268417

54A6;127D14;142 L8; 163O23;204P7

  

R: CTGTATTGCAGCTTCCGAGG

   

S074

Loc540077

F: CTGAATACCTGATCCGATGG

417

Exon

54A6;142 L8;163O23; 204P7

  

R: GCATGTGCATGAGTAGGTCC

   

S075

Loc786439

F: GGCGTCTTTAATCAGGATTTGG

200

UniSTS:222501

None

  

R: AATCCAACACTTGAAACCGACA

   

S076

ZNF318

F: CTGTCTTCACTCGAAGCTCC

438

ENSBTAT00000013481: Exon 1

24 L23;66 G8;83 N5; 119 J9;162 F10

  

R: AGCTCCTACTTCGTTCCTCC

   

S077

TJAP1

F: GAGGACGAGGAAGAGCTGAA

654

ENSBTAT00000035977: Exon 12

None

  

R: CGTGCAGAGGATTGAAGGAG

   

S078

POLH

F: GACAGCCACACACATAAGCA

497

ENSBTAT00000007900: Exon 11

68 F17;71 H18;74P6; 124 L6;250 J4

  

R: GTCTCACAGAGTCGGACACG

   

S079

MRPS18A

F: AGTCGTGAGACCACTGCAGC

191

ENSBTAT00000056429: Exon 6

115P10;176 M14; 233 H10;278 K6;291I13

  

R: AGGACCTCCTGAGAGCCTGA

   

S080

VEGFA

F: GATCATGCGGATCAAACCTCACC

326

UniSTS:471318

12B17;12 H11;30 L7; 63B18;124 J8;249D14

  

R: CCTCCGGACCCAAAGTGCTC

   

S081

MRPL14

F: TCAGAACTGCTCCATTCACG

182

UniSTS:64809

117 J15

  

R: CAACAACGTGGTCCTCATTG

   

S082

SLC29A1

F: GGTGGTCTTTGAGCACGACT

537

UniSTS:207086

None

  

R: CCGGAACAGGAAGGAGAAG

   

S083

AARS2

F: CACTGGAAGCACTGCTGACC

325

ENSBTAT00000018232: Exon 22

None

  

R: GCAGCCAGAACAGCCATGTA

   

S084

CDC5L

F: CCAACTCAGTGGAGGACCAT

750

UniSTS:267825

134E15;147I12

  

R: GGCTTTGTTTCTGGATTTGG

   

S085

SUPT3H

F: CTTCTGCCTGGAACTTGCACTTG

208

UniSTS:476839

23P23;80P15;110 F4;5;6

  

R: TGCTTACTGTCTCCCACCTAGATTG

   

S086

Loc536911

F: TACCAGCCACCGAGACCAA

309

UniSTS:280406

9 G19;9 H22;9I23;24; 59B8

  

R: AGAGGCTGTTTGACGCCATAG

   

S086b

CLIC5(BM1258)

F: GTATGTATTTTTCCCACCCTGC

158

UniSTS:56663

291I15

  

R: GAGTCAGACATGACTGAGCCTG

   

S087

ENPP4

F: GAACCAGCTCACCAATGTGT

595

ENSBTAT00000004547: Exon 2

72 M13;74O6;127 F7; 182 K12;299 N7

  

R: TCCTCTGCTTCACCACCTAA

   

S088

RCAN2

F: TCTTTACTGTCTGAGCCACC

132

UniSTS:69107

None

  

R: TACACTCAGAGCTAGTTTGC

   

S089

CYP39A1

F: AGGTGATGGTGGCAACTATG

200

UniSTS:15671

57E15;181B7;202D23; 213A17;261 M4

  

R: CATGTGTCCATAATTTGATTGC

   

S090

TDRD6

F: GAGTTCTTCCACCTGCCGTC

490

ENSBTAT00000013158: Exon 1

114B7;147E14;190 N9; 329 H12;350E16

  

R: ATACCTGAGCCATGCTCTCG

   

S091

Loc785478

F: TACGCCACCTACACACACAC

439

Exon

65 L20;133 M1;211 N8; 233B22;233O14

  

R: GACTGGTAGCTCCTGATCTG

   

S092

GPR116

F: CACATCCAGTGCTTATTCAT

302

ENSBTAT00000035930: Exon 18

291 M9

  

R: TAGACAGAGAAGTTGGCTTG

   

S093

GPR110

F: AGTGGACAGATACCGGCTGC

452

ENSBTAT00000028795: Exon 10

None

  

R: AGGTGTGGCCATGTGATGGA

   

S094

TNFRSF21

F: CAGAGCAGAAGGCACCAAGT

500

ENSBTAT00000047874: Exon 11

118P16;351 H10

  

R: ATTGTCTGCCTCCTTGGTCC

   

S095

LOC785024

F: GGTTGTCAAGCCACTCGAAT

611

Exon

14B7;79 L8;168 N8; 264 L6

  

R: CGGAGTATATGGCCAGTGTT

   

S096

LOC512926

F: AGAGCAGAAGGCACCAAGTC

437

Exon

27A8;290 J19;351 H10

  

R: ACGCTCTGCATCTCATCACA

   

S097

CD2AP

F: TACCACAACACCAACTGCAT

309

UniSTS:278169

1 H10;14A2;75 J19; 114B12;151 J21;166 L22

  

R: TTACCGGGATCACAGAAACA

   

S098

GPR115

F: CACAGTGGTGGCAGCAATAA

490

ENSBTAT00000003815: Exon 5

None

  

R: GAATAGAGTGCAATGCCGGT

   

S099

OPN5

F: CTACATCTGCCTGGCGGTCA

287

ENSBTAT00000021933: Exon 4

167I8;228 M7

  

R: CATGGCTGCTATGGATCCGA

   

S100

MGC148542

F: ACATTTTCTCCTTCTTTGGCTCC

272

UniSTS:133880

1A19;1B9;140A1; 216D18;319I16

  

R: GATAGAGGATGACGACAAATGGC

   

S101

Loc785693

F: AGCCAGGTAGAGTTCCAATG

518

Exon

17 K13;75E1;76B22; 103 F21

  

R: AGTCTCGGCAGTTACCTTGA

   

S102

MUT

F: AGCAAAGCACATGCCAAAAT

750

UniSTS:279392

74 J7;8;86P12;252B10; 255 G2;266O16;313 L2

  

R: TTCCCCAGAAGAAAGACAAC

   

S103

Loc787783

F: GGAATCATCAACCCAGTGAGAAAGC

269

UniSTS:476844

255 G2;266O16;274D6; 288I23

  

R: CACACGGCGGCAGAAAGAGG

   

S104

RHAG

F: GAATCGATGACCATCCATGC

470

ENSBTAT00000015012: Exon 4,5

53D7;173C22;186 L10; 226 G3;4;226 H7

  

R: AGAAGGCTGGAACATGCGTA

   

S105

Loc100138627

F: AATGAATAGTATCCCCAATACCTGC

150

UniSTS:164033

None

  

R: GTCCACAAAACATTCTCCTTTCC

   

S106

TFAP2D

F: TAAGCTTTCGGAGAAACCCA

1422

UniSTS:482175

5 K4; 139 L18;230 K5

  

R: CAGCAGCAAGACTCTCTGGA

   

S107

TFAP2B

F: TGCATGCTCCCTCCTCTC

120

UniSTS:71657

25D11;25 F24;142E22; 161A23;167 J23;189D14

  

R: CCTCGTCCAATTATGGTGCT

   

S108

Loc100138859

F: GGAGCACCACAGTACGTAAG

561

Exon

None

  

R: GAGGTGTGCCTGTATTGCTA

   

S109

Loc537895

F: TTCTCTCAAATGATGAATATGCTTC

270

UniSTS:251053

56 J7;86O3;87 H23; 277 G10;277 H11

  

R: GGACTATTCTATGCATGCCTCTC

   

S110

IL17A

F: CACTCAGGCTGTATCAATGC

591

ENSBTAT00000002786: Exon 3

13B24;74A7;74E17; 164 H22;164I23

  

R: CAGCTGTGTCATGTACTCCA

   

S111

MCM3

F: TGTCCCGATTTGACCTTCTC

515

UniSTS:268664

69 G8;168E20;223C7; 263 M23;270P6

  

R: GTCATCAGGGCTGAAGTTGG

   

S112

PAQR8

F: TCTATGTCCTGTCCTCCATC

447

ENSBTAT00000035844: Exon 2

102 M1;160 L10

  

R: AGAAGAAGTAGGCACTGACC

   

S113

TRAM2

F: TGTTCTACATCTTCATCGCCA

630

UniSTS:267311

13P23;53 J18;92C23

  

R: ACCAGATCACCGAGCTGAGA

   

S114

TMEM14A

F: CTACCCAAGAAACACTGTCGC

286

ENSBTAT00000006857: Exon 6

2C18;31C1;139B24; 183A23;280 K17

  

R: AGAGCATTCTATGAAGCCCG

   

S115

ICK

F: ACGGACTGGATCGCTAAGTA

627

ENSBTAT00000020711: Exon 14

2C18;76A8;77 G6; 198C12;199 K7

  

R: CAGAACAGCACAGCGGTATT

   

S116

GCM1

F: AGCTGTCCAACTGCCTCCTG

363

ENSBTAT00000010709: Exon 6

141A15;199 K7;230E24; 314I2

  

R: TGGGAAGGGGAGAAGTCGTA

   

S117

ELOVL5

F: CTACAGCCACGAGACAGTTT

182

UniSTS:279336

64 N21;82O21;90C20; 127 J19;163 F13

  

R: GGTTTCAATCATTCTTTCAT

   

* The bovine oligo primers were designed along the target bovine genomic sequence at an interval of ~80-160 kb between the two neighbor loci, depending on the availability of the DNA sequence that meet the primer selection criteria. A total of 119 pairs of primers were listed here.

BAC library organization and screening

To facilitate large scale PCR screening, all the 190,500 clones of the BAC library were organized into 3-dimensional BAC clone pools of plates, rows, and columns. Random BAC clones from each of 496 permanent 384-well storage plates were duplicated onto a Luria-Bertani (LB) agar plate for overnight growth at 37°C, using a 384-pin Multi-Blot Replicator as tool for BAC clone duplication (V & P Scientific, Inc., San Diego, CA). The overnight E. coli colonies were then harvested and pooled for plate (n = 496), row (n = 16), or column (n = 24). The standard alkaline lyses methodology was adapted for isolation of the pooled BAC plasmid DNA and the resulting DNA was assembled into super plates for routine PCR screening [35]. The first dimension of the BAC clone pool consisted of 496 DNA samples, each representing one of 496 BAC plates (P001-P496). The second and third dimension consisted of 16 and 24 DNA samples, respectively, for the pooled 16 rows (R01-R16) and 24 columns (C01-C24) of the random BAC clones.

To screen the BAC library using each of 119 pairs of comparative oligo primer pairs, the diluted DNA from each well of the super pool plates was used as a DNA template. The individual PCR reaction was adapted in a total of 10 μl reaction volume with 50 μM of dNTPs, 1.5 mM Mg++, 0.2 μM of each primer pair, 1 × PCR buffer, and 0.1 unit of Tag DNA polymerase. The PCR products were resolved by 1.5% agarose gel electrophoresis and the specific PCR fragment band with the expected size indicated a potential positive BAC clone for the gene loci of oligo primers used. The exact location of the target clone in the BAC library was determined by sequential PCRs using the super row and super column DNA as templates, respectively.

DNA fingerprinting and contig assembling

DNA fingerprinting was performed to determine the overlapping relationship among the identified positive BAC clones [12]. DNA from the positive BAC clone was purified from host E. coli by QIAGEN column and subjected for complete restriction enzyme digestion using Hin dIII. The enzyme digested products were analyzed on 1% TAE agarose gel electrophoresis for recoding of DNA fragment patterns. The fingerprinting images were captured with UVP Labworks System (UVP Inc., Upland, CA) for systematic analysis. Restriction fragment patterns were analyzed to identify overlapping BAC clones, which were then manually assembled into draft contigs based on the modified methods of Marra [36] and Soderlund [37].

BAC-end sequencing

BAC-end sequencing was performed for the selected clones to facilitate verification of the overlapping relationships of the BAC clones. The sequencing was performed on an ABI 3730X DNA analyzer at the core facilities of the Institute of Genetics and Developmental Biology, the Chinese Academy of Sciences. The oligo nucleotide primers used for the DNA sequencing were Copycontrol pCC1BAC vector-derived sequencing primer T7 (5’-TAATACGACTCACTATAGGG3’), pCC1/pEpiFOS RP-2 (abbr. RP2) (5’-TACGCCAAGCTATTTAGGTGAGA-3’), and pCC1/pEpiFOS RP-1(abbr. RP-1) (5'-CTCGTATGTTGTGTGGAATTGTGAGC-3'). The resulting sequences were analyzed for overlapping, and used as templates for oligo primer design. Based on the sequence data generated by BAC-end sequencing, PCR primers (Additional file 1: Table S1) were designed to amplify the common genetic loci in two overlapped BACs for confirmation. Sequence-Specific PCRs (SP-PCRs) were performed in 20 μl system including approximately 2 ng BAC DNA, 0.5 U Taq DNA polymerase, 0.1 mM dNTPs, 1.5 mM Mg++, 0.25 μM each primer, and 1× PCR buffer. When necessary, the PCR products were verified by cloning the fragments into a TA vector for verifying DNA sequencing.

Assemble of the BAC clone contig

A continuous BAC clone contig was eventually assembled based on the integrated results of DNA fingerprinting, BAC-end sequencing, and sequence specific PCR amplification of the common loci on the overlapping clones. Redundant BAC clones were removed from the assembly based on the necessity and the relative contribution of each overlapping BACs on the contig. Gaps in the contig were closed by the repeated cycles of PCR screening of BAC clones, DNA fingerprinting of additional BAC clones identified, BAC-end sequencing, and SP-PCR verification. Additional effort was made to link the existing BAC clone contig to the physical map constructed previously, for a complete physical map covering the entire ovine MHC including the autosome insertion between class IIa and IIb.

For comparison of the MHC structure and organization between sheep and other mammals, multiple comparisons were performed for the representative MHC and extended DNA sequences from human, chimpanzees, mouse, cattle, and sheep. Sequence data were downloaded from the NCBI database and other related public websites designated for the sheep genomic information.

Results

Target BAC identification

We successfully identified a total of 368 positive BAC clones for ovine chromosome 20 between MHC class IIa and IIb, utilizing bovine primers designed from the consensus genome region (Table1). Out of 119 pairs of oligo primers designed, 92 pairs worked effectively to generate specific target gene fragments of the expected sizes. This approach resulted in the successful identification of positive ovine BAC clones in the target genome region, and the overall efficiency of comparative PCR reached 80%. The relatively high rate of success for the comparative SP-PCR not only facilitated our mapping efforts, but also helped to confirm the homologous nature of MHC regions between bovine and ovine species.

Organization of ~190,500 random ovine BAC clones into three dimensional super DNA pool of rows (n = 16), columns (n = 24), and plates (n = 496) significantly increased the efficiency of PCR screening of the sheep BAC library (Figure1). The whole BAC library of 8.4× genome equivalents was screened through with a maximum of 536 (=496 + 16 + 24) PCR reactions, and a positive BAC clone could be frequently identified by as few as 136 (=96 + 16 + 24) PCR reactions using the super pool DNA as templates. In addition, PCR-based BAC clone screening also helped to eliminate the need for hybridization-based screening using radioactive 32P labeling.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-398/MediaObjects/12864_2012_Article_4266_Fig1_HTML.jpg
Figure 1

Representative gel images on initial PCR screening of an ovine BAC library using comparative primers from the bovine sequences. Approximately 190,500 random BAC clones were organized into pooled super DNA plates of rows, columns, and plates to facilitate PCR screening. Location of a target positive BAC clone in the library was determined usually by two runs of PCRs, one for “plate” and the other for “row + column”. The procedure eliminated the need for hybridization-based screening with radioactive 32P labeling. Gel images of PCR screen band on (A): Row pool of P098 BAC plate using the primer pair S036; (B): Row pool of P056 BAC plate using the primer pair S109; (C): Row N of P083 BAC plate; (D): Row F of P162 BAC plate. M: DL2000. Sample: PCR Products. A ~ P: Number of Row. 1 ~ 16: Number of Column (only partial shown here). P: Positive control (The amplified PCR products using the sheep genome DNA as templates).

DNA fingerprinting and BAC-end sequencing

The initial order of the positive BAC clones identified was successfully determined by inferring the overlapping relationships among the clones via DNA fingerprinting, using Hin dIII for restriction enzyme digestion of the BAC clone DNAs (Figure2). Out of 368 positive BAC clones subjected for the DNA fingerprinting, 185 clones with their overlapping relationships were successfully determined. The resulting BAC contig covered the entire autosome insertion region between the MHC class IIa and IIb. After removing the redundant clones, a total of 108 effective BACs were ordered to form an overlapping BAC contig (Additional file 1: Table S1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-398/MediaObjects/12864_2012_Article_4266_Fig2_HTML.jpg
Figure 2

A representative image of DNA fingerprints of the positive BAC clones for determination of overlapping relationship. The positive BAC clones identified in the previous steps were digested with Hin d III, followed by separation on a 1% agarose gel in 1× TAE buffer. The gel was stained with Ethidium Bromide (EB) for photograph with a UVP Labworks system. M: Marker of DNA size standard (1 kb plus DNA ladder from Invitrogen, San Diego, CA, USA) with the base pair (bp) sizes indicated on both sides.

For cross-checking of the clone order, BAC-end sequencing was performed for all overlapping BAC clones, and the sequences generated were used to design BAC-end oligo primers (Additional file 1: Table S1) for further verification of overlapping relationships. The sequences of 185 BAC-ends have been deposited into the NCBI database with the access number HR309252 through HR309068, corresponding to dbGSS ID 30164010 through 30163826.

Cross verification and physical map assembling

For additional cross-verification of the BAC clone orders, a total of 108 pairs of BAC-end oligo primers were designed for amplification by PCR of the common loci in two overlapping BACs (Figure3). Verification PCR confirmed the results of DNA fingerprinting at a high level of accuracy. Out of the 108 primer pairs used, 103 produced the specific PCR products with the expected size, the overall success rate reached 95% (Additional file 1: Table S1). An overlapping relationship between two BACs was further verified if the common target loci were detected from both BACs in the overlapped region. A total of five pairs of oligo primers failed to generate the specific PCR band, or failed to produce the PCR fragment at the expected size.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-398/MediaObjects/12864_2012_Article_4266_Fig3_HTML.jpg
Figure 3

PCR verification of the overlapping relationship between pairs of overlapping BAC clones. Pairs of overlapped BAC clones were PCR amplified using a primer pair designed based on the BAC-end sequence. The markers above the black lines define the primer pairs and the ones below the lines are numbers of positive clones used as PCR templates.

A complete physical map of a BAC clone contig for the ovine MHC region between class IIa and IIb was successfully assembled (Figure4), based on the integrated results of DNA fingerprinting, BAC-end sequencing, and confirmation PCR of the BAC ends. The fully assembled physical map was composed of 108 effective ovine BAC clones organized into a continuous contig that covered the entire region between ovine MHC class IIa and IIb (Figure4). Based on the results of DNA fingerprinting, no gaps exist in the constructed BAC clone physical map which spans approximately 14 Mb genome region of ovine chromosome 20, indicating the even distribution of BAC clones in the library we previously constructed.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-398/MediaObjects/12864_2012_Article_4266_Fig4_HTML.jpg
Figure 4

A 14 Mb BAC clone physical map covering the entire region between ovine MHC Class IIa and IIb. The order and orientation of BAC clones (overlapping horizontal bars with clone ID name listed above) were determined by combinations of DNA fingerprinting, BAC-end sequencing, and sequence-specific-PCR. Target gene identified by BAC-end sequencing is marked with a vertical bar along the horizontal line, with locus name listed above. The continuous BAC map is represented by three panels with the overlapping regions marked with the same colored shadows at the both ends.

Discussion

Using the comparative approaches, we successfully constructed a 14 Mb BAC clone contig map for a region in ovine chromosome 20 that harbors the MHC. Comparison between the identified ovine BAC contig and the orthologous bovine genomic region showed that the two species share essentially the same genomic structure and organization for the entire inversion/insertion between MHC class IIa and IIb (Figure5). For the available genetic loci generated via the SP-PCR and BAC-end sequencing, our results essentially confirmed the sheep genome sequence assembly presented by ISGC in the MHC region [33].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-398/MediaObjects/12864_2012_Article_4266_Fig5_HTML.jpg
Figure 5

Schematic presentation of MHC structures among representative mammal species. Bovine and ovine MHC is interrupted by a long piece of non-MHC insertion that divided class II into IIa and IIb subregions. The red, blue, and green color stands for MHC Class I, Class III, and Class II, respectively. The grey color gradient represents the extended Class II region. The order of loci in the extended Class II region of bovine and ovine is in an opposite orientation compared to that of human, chimpanzees, and mouse. Dash line marks the break point of a hypothetical chromosome inversion. Dashed circles indicate the hypothetical chromosome looping and the subsequent crossover occurred during the evolution of ruminants. The drawing is not to the scale.

The physical map of ovine BAC contig we constructed helped to provide additional evidence to support the hypothesis that, there was an ancient chromosome rearrangement in the ancestor of ruminants which shaped the MHC structures currently observed in the ovine and bovine (Figure5). It is obvious that the MHC region in human, mouse and chimpanzees is continuous with no interruption, but in bovine and ovine it is interrupted by a large piece of autosome insertion which divided MHC class II into IIa and IIb subregions (Figure5). Given the fact of opposite loci order and orientation for the insertion region in ovine and bovine relative to those of human and mouse, it is highly possible that an event of genetic recombination occurred to the ancestor chromosome of ruminants, probably via chromosome looping and the subsequent crossover. This possibility was suggested by researchers previously [29, 38].

Examination of the bovine DNA sequence from the public database showed that the total length of bovine MHC is ~20 Mb, including the extended Class IIb region [34]. However, the total length of the orthologous ovine MHC was ~14.3 Mb as determined in this study, which is approximately 5.7 Mb shorter than the MHC of bovine. On the other hand, the sequence of the same bovine region presented in the NCBI database is ~18 Mb in length (http://www.ncbi.nlm.nih.gov/projects/mapview/maps.cgi?taxid=9913&chr=23). These discrepancies may not likely be resolved unless highly accurate sequence maps for the entire MHC regions become available.

The reliability of the ovine BAC contig map reported here is sufficiently high in theory, partially due to the fact that the DNA fingerprinting was utilized to infer the BAC clone orders, plus the results were cross-verified by both of the BAC-end sequencing and SP-PCR amplification of the target loci. However, it is not escaped from our attention that there are 5 out of the 108 overlapping locations in the BAC map where the SP-PCR failed to generate the expected PCR products between the overlapping BAC clones (data not shown). The significance of such failure in relation to the overall quality of the map remains to be determined. The possible explanations include the error in SP-PCR primer sequences, the high level of heterogeneity or polymorphism of the target locus involved, or the mistake in the interpretation of results of DNA fingerprinting.

Combined with our previous BAC physical map for the ovine MHC, we have now assembled a completed BAC clone physical map with the inversion/insertion region included (Additional file 2: Figure S1). The physical map will help to generate an ovine MHC sequencing map with a high level of accuracy, which in turn will facilitate MHC functional studies and comparative MHC evolution studies in ruminants. DNA sequencing of the BACs is currently underway.

Conclusion

We constructed a high-density physical map for the sheep genome region between MHC class IIa and IIb via comparative approaches. A total of 108 effective ovine BAC clones were selected to form a continuous BAC contig that covers the entire non-MHC insertion. The map spans approximately 14 Mb in length, constituting ~25% of ovine chromosome 20. The entire ovine MHC region, including the autosome insertion for which the physical map has been constructed, is now fully covered by a continuous BAC clone contig. The accuracy of DNA sequences play vital roles in detailed SNP and other functional studies of MHC genes, as well as for genome evolution studies. The physical map will help to generate ovine MHC sequencing map with a high level of accuracy, which in turn will facilitate MHC functional studies, as well as the comparative MHC evolution in ruminants.

Notes

Declarations

Acknowledgements

The authors are very appreciative of the expert reviewers who helped to improve the quality of the manuscript significantly. This work was funded by research grants from National Natural Science Foundation of China (30125024; 30771148), Ministry of Science and Technology of China (2006DFB33750; 2010CB530204), and China Ministry of Agriculture (2009ZX08008-005B).

Authors’ Affiliations

(1)
School of Life Sciences, Shihezi University
(2)
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Science
(3)
Institute of Veterinary Animal and Biomedical Sciences, Massey University
(4)
Joint Research Center for Sheep Breeding and Developmental Biology, IGDB-Massey University
(5)
Graduate University of Chinese Academy of Sciences
(6)
Institute of Genetics and Developmental Biology, Chinese Academy of Science

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© Li et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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