Tissue Antigens ISSN 0001-2815

BRIEF COMMUNICATION

The diversity of major histocompatibility complex class II DRB1 gene in sheep breeds from Xinjiang, China M. Polat1,2,3 , Y. Aida2,3 , S.-N. Takeshima2,3 , J. Aniwashi4 & M. Halik1 1 College of Life Sciences and Technology, Xinjiang University, Urumqi, China 2 Viral Infectious Diseases Unit, RIKEN, Wako, Japan 3 Laboratory of Viral Infectious Diseases, Department of Medical Genome Sciences, Graduate School of Frontier Science, The University of Tokyo, Wako, Japan 4 College of Animal Science, Xinjiang Agricultural University, Urumqi, China

Key words Altai Argali wild sheep; Bashibai sheep; Karakul Ram; OLA-DRB1; phylogenetic tree Correspondence Dr Yoka Aida Viral infectious Diseases Unit RIKEN 2-1 Hirosawa Wako Saitama 351-0198, Japan Tel: +81 48 462 4408 Fax: +81 48 462 4399 e-mail: [email protected] and Dr Mahmut Halik College of Life Sciences and Technology Xinjiang University Urumqi 830046, China Tel: +86 991 8588001 Fax: +86 991 8588001 e-mail: [email protected]

Abstract Exon 2 of the ovine leukocyte antigen OLA-DRB1 locus was examined in sheep from the Xinjiang Karakul Ram and Bashibai populations, and three generations of hybrids were derived from a cross between Bashibai and Altai Argali wild sheep. This identified 12 novel alleles and 30 previously reported alleles. A neighbor-joining tree of the amino acid sequences of these 42 alleles revealed allelic clusters shared across the study populations. There were significant differences in allelic frequency between Karakul Ram and Bashibai sheep. DRB1*K18cC was the most frequent allele in Kararul Ram with a frequency of 21.2%, while DRB1*2F10c8 (13.2%) and DRB1*0803 (13.2%) were the most frequent alleles found in Bashibai sheep; the alleles DRB1*2F16c2, DRB1*1601, and DRB1*0803 occurred most frequently in F1, F2, and F3 populations, with frequencies of 17.6%, 14.3%, and 20%, respectively. Although many alleles were shared by Bashibai and hybrid sheep, some alleles differed between them, especially in the F1 generation of the Bashibai × Altai Argali cross. The hybrid-specific alleles indicated the introgression of Altai Argali alleles into hybrid flocks. A population tree based on the OLA-DRB1 allelic frequency in each population indicated that the Bashibai sheep and three hybrid populations were similar, with Karakul Ram being genetically distinct.

Received 11 March 2014; revised 14 October 2014; accepted 28 October 2014 doi: 10.1111/tan.12480

The major histocompatibility complex (MHC) is a family of highly polymorphic genes found in jawed vertebrates (1, 2). The MHC locus of sheep is known as the ovine leukocyte antigen (OLA) (3) and is located on chromosome 20 between bands q15 and q23 (4–6). The OLA class II genes encode a polymorphic glycoprotein composed of noncovalently linked α- and β-subunits. They play a pivotal role in the initiation of the immune response to pathogen-derived peptide antigens (7, 8). The DRB locus is the most polymorphic of the MHC class II genes (9), and thus OLA-DRB1 is highly polymorphic (10–13). Sequencing exon 2 from various breeds of sheep, including Latxa, Karrantzar, Finn, Russian Local, Romanov, Merino, Finnish Landrace, Texel, Coopworth, Suffolk, and Cheviot, has so far identified over than 89 OLA-DRB1 alleles (7, 11, 14–22). Previous studies have examined variation at the OLA-DRB1 locus in Chinese sheep breeds. Dongxiao and Yuan (23) studied the polymorphisms in exon 2 of MHC-DRB in Mongolian 50

and Kazakh sheep using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to define 18 genotypes controlled by seven alleles called A, B, C, D, E, F, and H. Li et al. (24) studied the association of MHC-DRB1 exon 2 genotypes with susceptibility or resistance to hydatidosis in three breeds of sheep, Chinese Merino (Sinkiang Junken type), Kazakh, and Doulang, and detected 14 alleles and 31 restriction patterns. Jian-ping et al. studied genetic variation in microsatellite sequences in introns of MHC class II DRB2, DYMS, and MB026, and DRB1 exons, genes in Small-tailed Han, Dorset, and Texel sheep, and showed rich genetic polymorphism at these four microsatellite loci in these breeds (25). Xinjiang, in the northwest of China, is one of the main areas of sheep breeding in the country, and nine indigenous, four improved, and four introduced sheep breeds are reared in this region (26). Most local sheep are not only adapted to the local environment but are also important genetic resources and one of © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 50–57

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the major components of agropastoral societies. The Bashibai sheep is one of the famous breeds of sheep native to Xinjiang. This breed is characterized by its large body size and strong resistance to disease and is reared for meat and wool (27). To decrease the size of its fat-tail and to increase meat productivity, veterinarians inseminated Bashibai ewes with sperm of the Altai Argali wild sheep, which is the largest of all sheep and has the shortest tail of any wild goat-antelope or sheep, to produce the first hybrid generation (F1). Backcrosses between F1 animals and Bashibai sheep produced the second (F2) and third (F3) generation hybrids (28). The Karakul Ram (also known as the Xinjiang Karakul Ram) is an improved breed derived from crossing the native Kucha sheep first with Karakul sheep from the former Soviet Union and then again with Kazakh and Mongolian sheep (29). The Karakul Ram is characterized by particularly strong resistance to harsh conditions. It is a multi-purpose breed kept for milking, pelts, and wool and has a distinctive meat. Its fiber is relatively coarse and is used for outer garments, carpets, and for felting (29). Both Karakul Ram and Bashibai sheep affect the quality of life of herdsmen and other citizens. The MHC variability in natural populations is of great interest to evolutionary biologists because of its high levels of polymorphism. Local sheep breeds are important for biological diversity and concerns about the conservation of genetic diversity have been expressed since the late 1990s (30). A deeper knowledge of the genetic diversity is essential for conservation decisions on maintenance and/or improvement. Karakul Ram and Bashibai sheep are the most important economical breeds in Xinjiang, but both sheep are threatened by multiple confounding factors. Although Xinjiang native sheep have been characterized using molecular markers such as mitochondrial D-loop (31) and microsatellite analysis (32), there have been very few studies of the MHC diversity in such sheep (24) and none addressing variation at this locus in the Karakul Ram and Bashibai breeds. This study was designed, therefore, to analyze genetic diversity in exon 2 of OLA-DRB1 from Xinjiang Karakul Ram, Bashibai, and three generations of hybrid (F1, F2, and F3) sheep by PCR-clone sequencing. This is the first study to characterize such variation in these breeds and populations. We collected blood samples from 116 animals: 83 sheep from Yumin County of Chochek city and 33 sheep from Kucha County of Aksu city, in Xinjiang Uyghur Autonomous Region. Genomic DNA was extracted from blood using the standard phenol–chloroform method (33). Next, the OLA-DRB1 exon 2 was amplified from all samples using PCR with primers DRB1.1 (5′ -TATCCCGTCTCTGCAGCACATTTC-3′ ) and DRB1.2 (5′ -TCGCCGCTGCACACTGAAACTCTC-3′ ), as previously described (34). The purified PCR products were legated into the vector pMD18T (Takara Bio-Engineering Co., Ltd., Dalian, China) and transformed into Escherichia coli DH5a. Eight clones were selected from each transformation and sequenced in both directions using the BigDye DNA © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 50–57

MHC-DRB1 diversity in Xinjiang native sheep

Sequencing Kit 2.0 (Applied Biosystems, Foster City, CA). For the novel alleles, the cloning was repeated using an independent PCR product. At least three clones were used for each alleles. Sequence data were analyzed using ASSIGN 400 ATF version 1.0.2.41 software (Conexio Genomics, Fremantle, Australia). Allele frequencies were obtained by direct counting: samples containing a single allele were considered homozygous, and thus that allele was counted twice. A 237 base-pair nucleotide partial sequence of OLA-DRB1 exon 2 were obtained for each allele. As these sequences are missing two polymorphic sites at the 3-prime end of the exon 2, (http://www.ebi.ac.uk/ipd/mhc/ovar/nomen_rules.html), we were unable at this stage to get official names for the novel alleles identified here. A phylogenetic tree was constructed from the deduced amino acid sequences of the 42 alleles present in these sheep, together with 89 previously reported alleles recorded in the IPD-MHC database (http://www.ebi.ac.uk/cgi-bin/ipd/mhc/ view_nomenclature.cgi?ovar.drb1) (Figure 1). This study found 30 previously known alleles and 12 novel alleles (OLA-DRB1*K18cC, *K26c1, *K67c11, *K6cE, *K68cB1, *1F11ch, *K48, *1F21c3, *3F7cB,*2F16c2, *2F10c8, and *3F6c3). The nucleotide sequences and deduced amino acid sequences of the 12 newly identified alleles were 84.4%–97.9% identical at the nucleotide level (Figure 2A) and 69.6%–96.2% identical at the amino acid level (Figure 2B) to the DRB1*0101 exon 2 sequence (accession number: FN543113). Furthermore, this tree clearly showed that there was no typical clade for the two breeds and three hybrid sheep populations. Alleles isolated from one population were shared by others. Low bootstrap values indicated the shallow divergence between populations (Figure 1). As shown in Table 1, we compared the frequencies of OLA-DRB1 alleles in Karakul Ram, Bashibai sheep, three hybrid generations derived from a Bashibai × Altai Argali cross and the previously reported allele frequencies for OLA-DRB1 to those found in Scottish Blackface sheep, in crosses between British Milk × Suffolk or British Milk × Texel (35), and in the Columbia, Rambouillet, and Polypay breeds (36). We found 24 alleles in the Karakul Ram population (n = 33), 14 previously reported and 10 novel alleles. In Bashibai sheep (n = 34), we found 22 alleles, 17 of which have been previously reported and 5 of which are novel (Table 1). Moreover, our analysis of three generations derived from the Bashibai × Altai Argali cross found a total of 17 alleles (11 previously reported and 6 novel) in F1 sheep (n = 18), 19 alleles (15 previously reported and 4 novel) in F2 sheep (n = 21) and 11 alleles (7 previously reported and 4 novel) in F3 sheep (n = 10) (Table 1). Thus, there were fewer OLA-DRB1 alleles in the hybrids than in Bashibai sheep; this may be due to the smaller numbers of sheep from the three hybrid generations available for this study. The Karakul Ram population was characterized by a high frequency of a novel allele, DRB1*K18cC, which was present 51

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Figure 1 Neighbor-joining tree constructed from the predicted amino acid sequences of the OLA-DRB1 exon 2 alleles from two sheep breeds, Karakul Ram and Bashibai, and three generations of Bashibai × Altai Argali hybrids, together with 89 previously reported alleles recorded in the IPD-MHC database. A phylogenetic tree was constructed using the amino acid sequences of the 42 alleles present in the sheep in our study, together with 89 previously reported alleles, based on a distance matrix with the neighbor-joining method of Saitou and Nei (38) in MEGA version 5 (39). Numbers are bootstrap percentages; bootstrapping was performed with 1000 replicates to assess the reliability of individual branches. The nucleotide sequences reported in this paper have been submitted to the International Nucleotide Sequence Database and have been assigned accession numbers: AB796309 (DRB1*K68cB1), AB796311 (DRB1*K6cE), AB796313 (DRB1*1F11ch), AB796314 (DRB1*K67c11), AB796316 (DRB1*K26c1), AB796317 (DRB1*K48), AB796322 (DRB1*1F21c3), AB796327 (DRB1*3F7cB), AB796328 (DRB1*2F16c2), AB796329 (DRB1*2F10c8), B796330 (DRB1*K18c), and AB796331 (DRB1*3F6c3).

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MHC-DRB1 diversity in Xinjiang native sheep

Figure 2 Alignment of (A) nucleotide and (B) predicted amino acid sequences of the β domains encoded by 12 new Ovar-DRB1 alleles. Alignment of the nucleotide (A) and predicted amino acid (B) sequences of the β domains encoded by 12 new Ovar-DRB1 alleles from the Karakul Ram or Bashibai breeds, or from Bashibai × Altai Argali hybrids. Nucleotides and amino acid residues identical to those encoded by Ovar-DRB1*0101 (accession number: FN543113) are indicated by dots; homology scores also refer to this allele. Identity is shown as a percentage.

in 21.2% of Karakul Ram individuals but absent from Bashibai sheep and the hybrids. The Bashibai breed contained high frequencies of DRB1*2F10c8 (13.2%) and DRB1*0803 (13.2%) alleles, both of which were relatively rare in Karakul Ram, F1, and F2 generations. However, both alleles were more common in the F3 generation, with DRB1*0803 being the most frequent (present in 20%) and DRB1*2F10c8 the third most frequent allele (present in 10%) in F3 sheep. DRB1*2F16c2 was present at a high frequency in the F1 generation (17.6%). Although this allele was present at a low frequency in both Karakul Ram and Bashibai breeds, it was © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 50–57

found at a relatively high frequency (10%) in the F3 population. The F2 generation was characterized by a high frequency (14.3%) of DRB1*1601; this allele was found in all the populations studied and had the second highest frequency (15%) in the F3 hybrid generation. DRB1*0803 occurred with a high frequency (20.0%) in the F3 generation, although this allele was found in all the breeds studied. Of the 42 alleles found in our study, nine (OLA-DRB1*0311, *1006, *1608, *K18cC, *K26c1, *K67c11, *K68cB1, *K48, and *K6cE) were found only in the Karakul Ram population, one (OLA-DRB1*0503) only in Bashibai sheep, one 53

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Table 1 Frequencies (%) of OLA-DRB1 alleles for the sheep breeds studieda Frequencies (%) Allele *1F11chd *1F21c3d *2F10c8d *2F16c2d *3F6c3d *3F7cBd *0101 *0102 *0103 *0201 *0303 *0311 *0401 *0402 *0405 *0502 *0503 *0701 *0803 *0806 *0901 *1001 *1006 *1202 *1301 *1302 *1303 *1403 *1502 *1601 *1604 *1607 *1608 *1801 *2001 *2002 *K18cCd *K26c1d *K48d *K67c11d *K68c1d *K6ced No. of Homf

Karakul (N = 33)

Bashibai (N = 34)

F1 (N = 17)

F2 (N = 21)

F3 (N = 10)

SBFb (N = 64)

BrM/SXb (N = 20)

BrM/Xb (N = 16)

Clbc (N = 129)

Rblc (N = 128)

Poyc (N = 126)

0.0 3.0 3.0 1.5 0.0 3.0 7.6 0.0 1.5 1.5 3.0 6.1 0.0 0.0 0.0 0.0 0.0 0.0 6.1 0.0 4.5 3.0 3.0 0.0 0.0 1.5 3.0 0.0 0.0 9.1 0.0 0.0 6.1 1.5 0.0 0.0 21.2e 3.0 1.5 3.0 1.5 1.5 7

0.0 4.4 13.2e 4.4 11.8 1.5 1.5 5.9 1.5 0.0 1.5 0.0 1.5 0.0 2.9 0.0 1.5 2.9 13.2e 2.9 0.0 1.5 0.0 0.0 2.9 0.0 1.5 0.0 0.0 11.8 0.0 2.9 0.0 7.4 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7

5.9 5.9 2.9 17.6e 8.8 11.8 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 2.9 2.9 2.9 8.8 0.0 0.0 0.0 0.0 2.9 0.0 0.0 2.9 0.0 5.9 0.0 0.0 5.9 5.9 0.0 0.0 0.0 0.0 0.0 0.0 3

0.0 0.0 4.8 7.1 11.9 4.8 0.0 0.0 0.0 0.0 2.4 0.0 0.0 7.1 4.8 0.0 0.0 4.8 2.4 0.0 0.0 2.4 0.0 2.4 0.0 2.4 2.4 0.0 9.5 14.3e 2.4 2.4 0.0 0.0 7.1 4.8 0.0 0.0 0.0 0.0 0.0 0.0 4

0.0 0.0 10.0 10.0 10.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 0.0 5.0 0.0 5.0 20.0e 0.0 0.0 0.0 0.0 0.0 0.0 5.0 0.0 5.0 0.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4

0.0 0.0 0.0 0.0 0.0 0.0 21.7e 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 9.2 0.0 0.0 15.8 0.8 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

0.0 0.0 0.0 0.0 0.0 0.0 2.5 20.0e 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.5 0.0 0.0 0.0 5.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

0.0 0.0 0.0 0.0 0.0 0.0 0.0 12.5 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9.4 25.0e 0.0 0.0 3.1 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 22.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.1 0.0 0.0 0.0 0.0 12.7e 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 11.6 1.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 —

BrM/TX, British Milk/Texel cross; BrM/SX, British Milk/Suffolk cross; Clb, Columbia breed; Poy, Polypay; Rbl, Rambouillet breed; SBF, Scothish Blackface. a All alleles found in this study are underlined and other alleles are refereed from previous study. b see Ref. (36). c see Ref. (37). d New alleles italicized. e The most frequent alleles given in bold face. f Number of homozygous individuals.

(OLA-DRB1*1F11ch) only in Bashibai × Altai Argali F1 hybrid, two (OLA-DRB1*1502 and *1604) only in the F2 population, and two (OLA-DRB1*1403 and *0502) only in the F3 population. By contrast, five alleles (OLA-DRB1*2F10c8, *2F16c2, *3F7cB, *0803, and *1601) were present in all five 54

populations. Furthermore, six alleles (OLA-DRB1*1F21c3, *0101, *0103, *0303, *1001, and *1801) were commonly found in both Karakul Ram and Bashibai breeds, whereas two alleles (OLA-DRB1*0901 and *1302) were present in Karakul Ram and one or more of the hybrid populations © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 50–57

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and also in other previous studies. On the other hand, six alleles (Ovar-DRB1*1F21c3, *3F6c3, *0405, *0806, *1607, and *2001) were common to the Bashibai population and the hybrids derived from it, with two further alleles (Ovar-DRB1*0402 and *2002) being found in the hybrid populations. The most interesting finding of this study was that six alleles, namely DRB1*1F11ch, *0502, *1403, *1502, *1604, and *2002, were found in the hybrids but not in Bashibai sheep, their maternal ancestor, or the other studied breeds, indicating these alleles may derive from the wild Altai Argali sheep. Table 1 also shows that all the sheep breeds from Xinjiang differed significantly from Scottish Blackface sheep, British Milk × Suffolk, and British Milk × Texel crosses, and the Columbia, Rambouillet, and Polypay breeds, not only in their allele frequencies but also in which alleles were present. Most of the 42 alleles found in Xinjiang sheep populations were not detected in these six populations. To investigate the evolutionary distance between the five different populations, we used neighbor-joining algorithms to construct a population tree based on the frequencies of OLA-DRB1 alleles in Karakul Ram, Bashibai sheep, three hybrid generations derived from a Bashibai × Altai Argali cross, and the allele frequencies reported previously for OLA-DRB1 in Scottish Blackface sheep, British Milk × Suffolk and British Milk × Texel crosses, and the Columbia, Rambouillet, and Polypay breeds (Figure 3). The population tree revealed clear topologies consistent with the historical and geographical origin of the breeds. Bashibai sheep plus its hybrids and Karakul Ram formed two separate major branches. Scottish Blackface, British Milk × Suffolk, and British Milk × Texel together formed one main cluster, and Columbia, Rambouillet, and Polypay sheep clustered into a different branch, indicating these breeds are genetically distinct from the others tested (Figure 3A). Bashibai sheep showed the smallest genetic distance from the hybrids derived from it. The genetic distances between Karakul Ram and Bashibai sheep and hybrids were smaller than those between Karakul Ram and geographically distant breeds and crossbreeds (Figure 3B). These significant differences may result from the historical breeding process, natural or artificial selection, effect of inbreeding, and the genetic origins of the Xinjiang sheep breeds and hybrids. This population tree also indicated the close relationship between Bashibai sheep and the hybrid populations. These results agreed with the breeding history of Bashibai sheep and the hybrid sheep populations (28). Our results support the hypothesis that the genetic diversity in the Xinjiang breeds is at least partially explained by their historical origin. We found the allele frequency of OLA-DRB1 locus differed significantly between Karakul Ram and Bashibai sheep, not only in the percentages of the most frequent alleles but also in the incidence of breed-specific alleles. Karakul Ram had greater diversity in OLA-DRB1 alleles than Bashibai sheep. Although Bashibai sheep and the derived hybrid populations © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Tissue Antigens, 2015, 85, 50–57

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Figure 3 Population tree of Karakul Ram, Bashibai, and three generations of Bashibai × Altai Argali hybrids, together with Scottish Blackface, British Milk × Suffolk, British Milk × Texel, Columbia, Rambouillet, and Polypay breeds. The population tree was constructed by POPTREE2 software (41) using the neighbor-joining algorithms (38) (A) from Nei’s genetic distances (B), which were calculated from allele frequencies (40) in the OLA-DRB1 gene in two breeds, Karakul Ram and Bashibai, and Bashibai × Altai Argali hybrids, and in the previously studied populations: Scottish Blackface (SBF), British Milk × Suffolk (BrM/S X), British Milk × Texel (BrM/T X), Columbia (Clb), Rambouillet (Rbl), and Polypay (Poy) breeds.

shared many alleles, differences were seen between these populations due to hybrid-specific alleles inherited paternally from wild Altai Argali. Therefore, to maintain MHC diversity in hybrid Bashibai sheep, it will be necessary to prevent the hybrids from crossing with other breeds and to abstain from further backcrossing with Bashibai sheep. Six alleles, DRB1*1F11ch, *0502, *1403, *1502, *1604, and *2002, were found in the hybrid sheep but not in their maternal ancestor, Bashibai sheep, or in other breeds. Samer Alasaad has identified paternal inheritance of the Capy-DRB1*5 allele from the Iberian ibex and demonstrated that variation at MHC loci may be used to detect hybridization between wild species and their domesticated relatives (37). These six alleles, especially the novel allele DRB1*1F11ch, might be Argali MHC-DRB1 alleles or alleles derived from Altai Argali. It is possible that detection of alleles paternally inherited from wild Altai Argali was limited by the small number of samples collected from hybrid sheep. Karakul Ram and Bashibai sheep populations contained similar numbers of homozygous individuals. However, as expected, the F1 hybrid generation showed reduced levels of DRB1 homozygosity compared with Bashibai sheep. The percentage 55

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of homozygote’s increased in F2 and F3 generations, probably as a result of backcrossing the hybrids to Bashibai sheep. This is the first report of allelic distribution in the OLADRB1 gene in Karakul Ram, Bashibai sheep, and hybrid populations from Xinjiang. We found a high rate of polymorphism and diversity in the OLA-DRB1 gene in Xinjiang sheep populations despite the relatively small sample sizes of each group. Characterizing the allelic distribution and diversity of this gene in these populations broadens our knowledge and understanding of variability at the sheep MHC locus, which is important for protection against pathogens and for the immune response. Our study provides new information, important both for animal husbandry and for the scientific design of breeding strategies to produce more resistant livestock for the future. Acknowledgments

We thank Professor Zhang Ya Ping, Zhou Chun Ling, and Wu Shi Fang (Kunming Institute of Zoology, CAS), Associate Professor Yuli (Yunnan University), and Liu Wei [Yaning Bioscience (Shenzhen) Co., Ltd] for supplying laboratory facilities. This study was supported by National Natural Science Foundation of China (No.31060152; 31360266), Funding of cooperation projects of Kunming Institute of Zoology, and Key Discipline Funding of Zoology, Xinjiang University. This work was supported by Grants-in-Aid for Scientific Research (B and C) from the Japan Society for the Promotion of Science (JSPS) and by a Grant of Program for the Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry from Japan. Conflict of interest

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The diversity of major histocompatibility complex class II DRB1 gene in sheep breeds from Xinjiang, China.

Exon 2 of the ovine leukocyte antigen OLA-DRB1 locus was examined in sheep from the Xinjiang Karakul Ram and Bashibai populations, and three generatio...
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