Allelic variations in exon 2 of caprine MHC class II DRB3 gene in Chinese indigenous goats

Small Ruminant Research 66 (2006) 236–243

Allelic variations in exon 2 of caprine MHC class II DRB3 gene in Chinese indigenous goats Meng-Hua Li a,c , Kui Li a,b , Juha Kantanen c , Zheng Feng a , Bin Fan a , Shu-Hong Zhao a,∗ a

Laboratory of Molecular Biology & Animal Breeding, College of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China b Department of Gene and Cell Engineering, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 10094, China c Animal Breeding, Animal Production Research, MTT Agrifood Research Finland, FIN-31600 Jokioinen, Finland Received 1 August 2005; received in revised form 2 September 2005; accepted 13 September 2005 Available online 10 November 2005

Abstract Allelic variations in the second exon of caprine leukocyte antigen-DRB3 gene (CLA-DRB3*02) were investigated. A total of 459 animals from 12 Chinese indigenous goat populations were genotyped. Six alleles and 18 restriction digestion profiles were distinguished by digestion of PCR amplification product of CLA-DRB3*02 with HaeIII. The results suggested that there were significant difference (P < 0.000) in both allele frequencies and genotype frequencies of exon 2 restriction fragment length polymorphisms (RFLPs) among the breeds. The new allele CLA-DRB3*0206 was identified only in three goat populations from the three-gorge reservoir, and the breed-specific allele CLA-DRB3*0205 was merely detected in the three populations of Tibetan goat. The results also indicated that the potentially different ecological factors, such as climate, disease, topography, pasture conditions and pathogens might be account for the significantly different distribution of alleles and genotypes among the goat populations. The nucleotide and predicted amino acid sequence alignment of CLA-DRB3*02 alleles provided evidence for considerable variation among the six alleles. Phylogenetic analysis showed that the six alleles of CLA-DRB3*02 were closely related. They clustered together and were separated from the corresponding sequences of sheep and cattle, which indicated the trans-species evolution model of CLA-DRB3*02 was not present in the Chinese goat populations studied. © 2005 Elsevier B.V. All rights reserved. Keywords: Chinese indigenous goat; DRB3 gene exon 2; Allelic variation

1. Introduction The major histocompatibility complex (MHC) of mammals includes two subfamilies of genes (MHC class I and MHC class II) whose products are cell surface glycoproteins that present peptides to T-cells. MHC class ∗ Corresponding author. Tel.: +86 27 87281306; fax: +86 27 87280408. E-mail address: [email protected] (S.-H. Zhao).

0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.09.017

I molecules are composed of two polypeptide chains: an ␣- and a ␤2 -microglobulin. MHC class II molecules, which are heterodimers composed of an ␣ and a ␤ chain, are expressed on the surface of antigen-presenting cells (Klein, 1986). While class I and II molecules appear somewhat structurally similar and both present antigen to T-cells, their functions are quite distinct. The major difference lies in that class I molecules present “endogenous” antigen to cytotoxic T-lymphocytes, while class II molecules play a critical role in the initiation of the

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immune response by presenting intracellularly processed peptides (exogenous antigens) to specially stimulate helper T-lymphocytes. A striking feature of MHC class II genes is the extensive polymorphism and this polymorphism is characterized by a large number of alleles at each locus and a large number of amino acid substitutions between alleles. The caprine lymphocyte antigen (CLA) system, e.g. the major histocompatibility complex of goat, has been shown to be similar to that of cattle which have two expressed class II antigens, DQ and DR (Takada et al., 1998). MHC molecules of DR subtype have been identified as one of the principle class II proteins found on the surface of goat cells (Schwaiger et al., 1993). So far, at least two DRB loci have been characterized (Schwaiger et al., 1993; Amills et al., 1995). The CLA-DRB3 exon 2 (CLA-DRB3*02) encodes the ␤1 domain of the DR molecule, which is in close contact with the foreign antigen and displays a very high degree of polymorphism with more than 25 different sequences identified to date. The extensive polymorphism of CLA-DRB3*02 is considered functionally to be responsible for the differences among individuals in the immune response to infectious agents. Associations of alleles of the bovine major histocompatibility complex DRB3 exon 2 (BoLA DRB3*02) with occurrence of disease and production traits have previously been documented (Sharif et al., 1998a,b). However, little is known about the associations between CLA-DRB3*02 alleles and the resistance to disease (e.g. Cowdriosis and nematode infection) and production traits (e.g. meat and milk) of goat. Chinese indigenous goat breeds are valuable resource in the world (Yue, 2000). Resulted from the long-term natural and artificial selection, Chinese indigenous goat breeds have formed some instinctive merits: extensive adaptability, high reproductive capacity, relatively rapid growth rate and strong coarse-fodder enduring ability. Especially Tibetan goat has the strong ability of resistance to certain diseases, frigidness and thin oxygen in Qinhai-Tibet plateau. Additionally, the goat populations from three-gorge reservoir survive in the very humid and hot environment. However, nowadays the population size of Chinese indigenous goat breeds is declining, which may be due to the introduction of exotic commercial goat breeds and the genetic improvement experiences (Guo, 2001). If the decline in population size continues, the genetic variation at these immunologically important genes will likely be lost, leaving these populations susceptible to outbreaks of infectious disease which would undoubtedly contribute to their future extinction (Garrigan and Hedrick, 2001). In this study, the genetic variations of CLA-DRB3*02 in 12 perilously

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endangered Chinese indigenous goat populations were investigated, not only to assay the amount of genetic variation presently retained in the goat populations, but also to direct future studies in the contribution of MHC alleles to susceptibility to disease caused by the infectious microorganisms. Several methods have been used to investigate the genetic polymorphism of MHC-DRB3 gene (Ledwidge et al., 2001). RFLP analysis of gene segments amplified by the PCR (Saiki et al., 1998) has been found useful for CLA-DRB3*02 typing (Amills et al., 1996). 2. Materials and methods 2.1. Animals and DNA preparation A total of 459 goats, which were randomly sampled and represented 12 Chinese indigenous goat populations, were analysed in this study. Sample size and locality for each population are listed in Table 2. Blood collection and genomic DNA preparation were conducted according to Li et al. (1997). 2.2. Molecular analysis Amplification of CLA-DRB3*02 was conducted by using primers DRB1.1 (5 -TATCCCGTCTCTGCAGCACATTTC-3 ) and DRB1.2 (5 -TCGCCGCTGCACACTGAAACTCTC-3 ) described by Amills et al. (1995). The DRB1.1 primer covers intron 1/exon 2 boundary and includes the last 18 bases of intron 1 and the first 6 bases of exon 2, while the DRB1.2 primer is complementary to the 3 end of exon 2. The PCR reaction was carried out in a final volume of 50 ␮l containing PCR buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.3, 1% Triton X-100), 1.5 mM MgCl2 , 100 ␮M of each dNTP, 2 ␮M of each primer, 2U of Taq polymerase (Promega, Madison, WI, USA) and approximately 300 ng genomic DNA. The amplification conditions comprised an initial denaturation of 95 ◦ C for 4 min followed by 30 cycles of denaturation at 95 ◦ C for 1 min, annealing at 63 ◦ C for 1 min and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 7 min performed in a Gene Amp PCR System 9600 (Applied Biosystems, Foster City, CA, USA). The 50 ␮l PCR amplification products were then concentrated into a total volume of 8 ␮l for the restriction enzyme digestion according to a procedure similar to that described by Sun (2001). Eight microlitres of concentrated PCR products were digested with HaeIII according to the manufacturer instructions. HaeIII restriction fragments were resolved

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by 8% polyacrylamide gel electrophoresis and were visualized with silver staining. PBR322/MspI (TAKARA, Japan) was used as a size marker for determination of restriction fragment size. Six CLA-DRB3*02 alleles (CLA-DRB3*0201, 168 bp/117 bp; CLA-DRB3*0202, 154 bp/14 bp/117 bp; CLA-DRB3*0203, 154 bp/14 bp/52 bp/65 bp; CLADRB3*0204, 154 bp/131 bp; CLA-DRB3*0205, 220 bp/ 65 bp; CLA-DRB3*0206, 40 bp/180 bp/65 bp), which determined the 18 restriction digestion profiles, were detected and confirmed by sequencing. Four polymorphic HaeIII sites were identified. The nucleotide sequences of six PCR products, which were homozygous at each of six CLA-DRB3*2 alleles, were determined by direct sequencing and confirmed by cloning into pGEM5 T vector (Promega, Madison, Wisconsin, USA) followed by sequencing. Sequencing was carried out on an ABI377 automated sequencer (Applied Biosystems, Foster City, CA, USA) in both directions and sequence was accepted if at least four of six reactions produced identical results at a given base.

2.3. Statistical analysis Frequencies of the six above-mentioned CLADRB3*02 alleles and the genotypes in 12 Chinese indigenous goat populations were calculated. Homogeneity tests of allele frequency and genotype frequency among all the populations were determined using Fisher’s exact test, which was implemented by SAS procedures (SAS Institute, Cary, NC, USA). The nucleotide and amino acid sequence analyses of CLA-DRB3*02 alleles were carried out using the DNAsis programs (Hitachi software Ltd., San Bruno, CA) for multiple alignment. Phylogenetic analysis was performed employing the MEGA2 software (Kumar et al., 2001) and the phylogenetic tree was constructed according to the neighbor-joining method (Saitou and Nei, 1987). This reliability of the dendrogram topology was determined by bootstrapping with 500 replicates (Felsenstein, 2000). The deduced amino acid sequences of the six CLA-DRB3*02 alleles detected in this study were combined with the cattle and sheep MHC-DRB3*02 amino acid sequences available from Takada et al. (1998). The number of non-synonymous substitutions per non-synonymous site (dN ) and the number of synonymous substitutions per synonymous site (dS ) were calculated using the method of Nei and Gojobori (1986) and were tested against the tdistribution with infinite degrees of freedom (Kumar et al., 1993).

3. Results 3.1. RFLP analysis The oligonucleotide primers DRB1.1 and DRB1.2 amplified the CLA-DRB3 exon 2 and gave a single product of the expected size (∼285 bp). RFLPs were detected with restriction digestion enzyme HaeIII. One novel allele was identified. It was resulted from the mutations of G, C and C at the positions 40, 41 and 42 of the amplified fragments, respectively, which created a HaeIII polymorphic site. Genotype frequencies and allele frequencies of CLA-DRB3*02 digested with HaeIII in 12 Chinese indigenous goat populations are described in Tables 1 and 2. As shown in the tables, the predominantly occurring alleles and genotypes were present in certain populations. On the other hand, no-specific PCR products and deficiency of allele were detected in some populations. These observations were supported by the similar cases present in the same samples by microsatellite analysis (Li et al., 2002). Additionally, the breed-specific allele CLA-DRB3*0205 was present only in the three populations of Tibetan goat (North Tibetan goat, East Tibetan goat and South-east Tibetan goat). The new allele CLA-DRB3*0206 was merely present in the three populations from the three-gorge reservoir (Chuandong White goat, Black goat and Nanjiang Brown goat). The distribution of allele frequency and genotype frequency among the total goat populations deviated significantly from the homogeneity expectation (P < 0.000), as determined by the Fisher’s exact tests. 3.2. Sequence analysis Among the 267 nucleotides and 89 amino acid positions, 40 (15.0%) nucleotide positions and 31 (34.8%) amino acid residues were variable. Many of the variations were localized to the specific regions. Four long polymorphic regions and three high variable regions (HVR) (Figs. 1 and 2) were found in the nucleotide and deduced amino acid sequences, respectively. The patterns of polymorphism in the six CLA-DRB3*02 alleles appeared to match those found in other species including human (Marsh et al., 2001), cattle (Davies et al., 1997) and sheep (Takada et al., 1998). Using the approximate method of Nei and Gojobori (1986), the number of nonsynonymous substitution per non-synonymous site (dN ) was 11.1 times higher than the number of synonymous substitutions per synonymous site (dS ) in the putative peptide-binding region (PBR). However, the corresponding ratio in the putative non-peptide-binding region (nonRBR) was not significant from the unity (Table 3).

6.06 9.08 9.08 0 15.2 9.08 0 15.2 3.03 15.1 6.06 9.08 3.03 0 0 0 0 0 0 0 12.8 0 5.0 15.4 7.7 18 10.3 5.1 7.7 7.7 10.3 0 0 0 0 0 13.9 22.2 11.0 0 8.3 22.2 2.8 5.6 5.6 0 2.8 2.8 2.8 0 0 0 0 0 22.7 13.6 59.1 0 4.5 0 0 0 0 0 0 0 0 0 0 0 0 0 17.7 8.8 14.7 0 5.9 14.7 2.9 5.9 0 0 0 0 0 5.9 8.8 5.9 5.9 2.9 6.3 6.3 40.6 0 3.1 6.3 0 9.4 3.10 0 0 0 0 9.4 3.1 0 9.4 3.0 3.7 7.4 18.5 0 29.6 7.4 0 7.4 0 0 0 0 0 3.7 14.8 0 3.7 3.7 9.09 31.8 22.7 0 4.5 27.4 0 4.5 0 0 0 0 0 0 0 0 0 0 DRB*0201/DRB*0201 DRB*0201/DRB*0202 DRB*0201/DRB*0203 DRB*0201/DRB*0204 DRB*0202/DRB*0202 DRB*0202/DRB*0203 DRB*0202/DRB*0204 DRB*0203/DRB*0203 DRB*0203/DRB*0204 DRB*0204/DRB*0204 DRB*0205/DRB*0205 DRB*0205/DRB*0201 DRB*0205/DRB*0202 DRB*0206/DRB*0206 DRB*0206/DRB*0201 DRB*0206/DRB*0202 DRB*0206/DRB*0203 DRB*0206/DRB*0204

0 0 0 3.4 0 44.8 3.4 27.6 20.7 0 0 0 0 0 0 0 0 0

0 0 22.2 0 11.1 16.7 11.1 33.3 5.6 0 0 0 0 0 0 0 0 0

0 0 8.8 21.7 0 0 13 17.4 13 26.1 0 0 0 0 0 0 0 0

0 0 80 0 0 0 0 0 0 0 0 0 0 0 4 4 4 8

Chuandong White goat Nanjiang Brown goat Small-xiang goat Matou goat Liaoning goat Neimonggol goat Taihang goat

Genotype frequencies (%)

Table 1 RFLPs genotype frequencies of exon 2 of CLA-DRB3 gene digested with HaeIII in 12 Chinese indigenous goat populations

Black goat

Wu goat

Southeast Tibetan goat

North Tibetan goat

East Tibetan goat

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Fig. 2 showed the amino acid alignment for the six CLA-DRB3*02 alleles, combined with OLA-DRB3*02 and BoLA-DRB3*02. Numbers referred to codon positions according to alignment with goat DRB sequences (Takada et al., 1998). Comparing the above-mentioned deduced amino acid sequences, we found that the Cysteine residue (position 15), as well as the glycosylation site (amino acid positions 19–21), was conserved. In addition, the codon positions 51–55 were similar to the amino sequence TELGR, which was found to be highly conserved in primate DRB genes. This position is located at the transition between ␤ sheet and the ␣ helix and is encoded by a nucleotide sequence, which is believed to promote recombination both between different alleles and between different loci (Gyllensten et al., 1991). Thirty-one amino acid replacements in CLADRB3*02 were found in aligning the deduced amino acid sequences. In particular, comparing our data with other goat DRB3*02 sequences (Schwaiger et al., 1993; Amills et al., 1995; Takada et al., 1998) indicated that eight new replacements have appeared at the positions 7 (Lys), 11 (Ala), 13 (Thr), 32 (Asn), 38 (Ala), 62 (His), 73 (Pro), 79(Tyr), respectively, while another 10 amino acid replacements described previously at the positions 9 (Arg), 11 (Ser), 12 (Thr), 13 (Ser), 57 (Ser, Glu, Asn), 67 (Leu), 70 (Asp), 77 (Lys), were not detected. Most amino acid variations in this region were thought to be functionally important for antigen recognition by forming the antigen-binding site (ABS) (Brown et al., 1993), although some variations were recognized in the conserved segment of CLA-DRB3*02. With the aim of testing the validity of trans-species model of evolution in the CLA-DRB3 region, the second exon of OLA-DRB3, BoLA-DRB3 and CLA-DRB3 alleles were subjected to phylogenetic analysis (Fig. 3). In the tree, alleles of CLA-DRB3*02 belonged to the same clade, and then clustered with OLA-DRB3*02 and BoLA-DRB3*02 successively. The accuracy of tree was supported by the bootstrap values. 4. Discussion In this study, the highly polymorphic nature of caprine DBR3 gene exon 2 has been demonstrated by RFLP and sequence analyses. The extensive polymorphism observed at caprine DRB3 locus shows the hallmark typical of classical MHC genes, i.e. (1) multiple nucleotide and amino acid substitutions between alleles, and (2) a large number of alleles (Snibson et al., 1998). Frequency distribution of both alleles and genotypes of CLADRB3*02 are deviated significantly from the homogeneity expectation (P < 0.000) among the goat populations.

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Table 2 Locality, number of animal sampled and allele frequency of exon 2 of CLA-DRB3 gene digested with HaeIII in12 Chinese indigenous goat populations Populations

Taihang goat Neimonggol goat Liaoning goat Matou goat Small-xiang goat Nanjiang Brown goat Chuandong White goat Black goat Wu goat Southeast Tibetan goat North Tibetan goat East Tibetan goat

Locality

Hebei province Neimonggol Autonomous Region Liaoning province Hubei province Guizhou province Three-gorge reservoir area Three-gorge reservoir area Three-gorge reservoir area Sichuan province Basu county, Tibet Nachu county, Tibet Nangkazi county, Tibet

No.

CLA-DRB3*02 alleles (%) DRB3*0201

DRB3*0202

DRB3*0203

DRB3*0204

DRB3*0205

DRB3*0206

37 27

1.8 11.1

24.1 25

60.3 55.6

13.8 8.3

0 0

0 0

27 26 28 35

15.2 36.4 42 24.1

6.5 34.1 12 37

28.3 29.5 42 22.2

50 0 4 1.9

0 0 0 0

0 0 0 14.8

36

31

36 35 58 57 57

9

39

4

0

17

33.1

22.1

23

2.8

0

19

59 31.8 10.2 19.7

11.4 33.2 21.3 25.8

29.6 25 37.3 25.8

0 4.2 14.1 16.6

0 5.6 16.7 12.1

0 0 0 0

It is tempting to speculate that this may reflect the difference in founder populations, selection pressures and/or the population sizes of goat breeds studied. Variations in frequency of alleles and genotypes are also observed within populations (Tables 1 and 2). This may be resulted from genetic drift and/or selection for production traits in the history of the populations (Sharif et al., 1998a). The newly identified allele, CLA-DRB3*0206, was unique to the populations from the three-gorge reservoir (Nanjiang Brown goat, Black goat and Chuandong White goat). Additionally, the breed-specific allele, CLA-DRB3*0205, was only found in the three populations of Tibetan goat (North Tibetan goat, East Tibetan goat and Southeast Tibetan goat). Given the importance of MHC genes in determining response to diseases, it could be hypothesized that the breed-specific allele has occurred through the natural selection. Further studies in associations between particular CLA-DRB3*02 alleles and Tibetan goat’s resistance to specific diseases are needed to confirm the assumption. An alternative expla-

nation for the common CLA-DRB3*02 alleles among some populations is that the common or similar microenvironments, such as the climate, ecological conditions and topographical features, may have exerted a large effect on it. A high genetic polymorphism was detected in CLA-DRB3*02, especially in the putative peptidebinding residues involved in antigen recognition. The observed polymorphic patterns corresponded well to those reported for MHC class II DRB region in other vertebrates, not only in human, but also in cattle and sheep. Analysis of DNA sequences of CLA-DRB3*02 had provided compelling evidence that, as firstly proposed by Doherty and Zinkernagel (1975), this polymorphism was maintained by a form of balancing selection (such as overdominant selection or “heterozygote advantage”). It is a fact that MHC heterozygotes have broader immune surveillance than homozygotes. There was also a significant excess of non-synonymous over synonymous nucleotide substitutions in the PBR codons of

Table 3 Number (S.E.) of non-synonymous substitutions per non-synonymous (dN ), number of synonymous substitutions per synonymous site (dS ), the ratio between dN and dS , results of t-tests, and number of codons in the peptide-binding region (PBR) and the non-PBR of CLA-DRB3*02 sequences in 12 Chinese indigenous goat populations dN PBR Non-PBR t ***

P < 0.001.

0.690 (0.025) 0.048 (0.019) 4.541***

dS 0.062 (0.035) 0.037 (0.017) −0.321

dN :dS

t

No. of codons

11.1 1.3

4.275***

20 69

0.134

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Fig. 1. An alignment of nucleotide sequences among the alleles of CLA-DRB3 exon 2 of 12 Chinese indigenous goat populations. Identity with the top sequence is indicated by dashes; the letters underlined refer to the polymorphic regions; the HaeIII digestion loci are indicated by shadow. Nucleotides are numbered according to that of human DRB3 gene.

CLA-DRB3*02 (dN :dS = 11.1), while non-synonymous and synonymous substitutions did not differ in the nonPBR (dN :ds = 1.3). These results strongly indicated that natural selection favoured novel PBR amino acid composition (Garrigan and Hedrick, 2001) and these findings were evidences that MHC polymorphism is selectively maintained and the basis of this selection is peptide binding and thus pathogen resistance (Hughes, 2000). In the long-term evolution, ancient and silent mutations also hitchhiked with translated mutations and became maintained in these regions (Simmons et al., 1994). In addition to the highly polymorphic regions, the conserved structure present in the CLA-DRB3*02 alleles in 12 Chinese indigenous goat populations suggested that there were antigens specific for infectious xenobiotics of goat. On the other hand, the high level of poly-

morphism found in PBR suggested that CLA-DRB3*02 corresponded to a wide range of antigens (Takada et al., 1998). The phylogenetic tree suggested that trans-species evolution had not occurred in the exon 2 of CLA-DRB3 gene in Chinese indigenous goat population. This indicated that most mutations of CLA-DRB3*02 in Chinese indigenous goat populations were not very ancient and were after speciation between goat and other ruminants. Crossing, microrecombination and point mutation were suggested to be account for the generation of new alleles. In addition, the genetic relationships of MHC-DRB3 exon 2 in goat, sheep and cattle showed by the phylogenetic tree were in accordance with the speciation of these species in the evolution history.

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Fig. 2. An alignment of the deduced amino acids sequences of six CLA-DRB3*02 alleles of 12 Chinese indigenous goat populations combined with that of OLA-DRB3*02 and BoLA-DRB3*02. A spiked circle indicates the conserved Cysteine residue; the potential site of the N-liked glycosylation position is underlined; the three high variable regions (HVRs) are shadowed. A dash indicates that the amino acid is the same as the top sequence; an asterisk indicates the position of a residue predicated to participate in the PBR. Codons are numbered according to alignment with goat, cattle and human DRB3 (Takada et al., 1998).

Fig. 3. Phylogenetic tree for the amino acid sequences of six CLA-DRB3*02 alleles in this study, plus previously reported OLA-DRB3*02 and BoLA-DRB3*02 sequence (Takada et al., 1998). Numbers on the branches indicate percentages of bootstrap values recovered after 500 replicates. Only the nodes supported by more than 50% of the 500 bootstrap replicates are labelled.

Despite the decline in population size of these Chinese indigenous goat populations, our results indicated that extensive MHC class II polymorphism had been retained in the remnant goat. Further studies in caprine MHC will be important, both for comparative studies and for understanding individual variation in response to vaccination, infection and disease in goat (Kenndey et al., 2002). Some measures should also be adopted to prevent the future extinction of endangered Chinese indigenous goat breeds.

Animal Breeding at Huazhong Agricultural University (Wuhan, PR China). The work was supported by the IFS (International Foundation for Science) Project (B/25802) and the National Natural Science Foundation of China to Dr. Shu-Hong Zhao, the National Outstanding Youth Science Foundation of China (39925027) and the Key Project of National Basic Research and Development Plan of China (G2000016103) to Dr. Kui Li.

Acknowledgements

Amills, M., Francino, O., S`anchez, A., 1995. Nested PCR allows the characterization of Taq I and PstI RFLPs in the second exon of the caprine MHC class II DRB gene. Vet. Immunol. Immunop. 48, 313–321. Amills, M., Francino, O., S`anchez, A., 1996. A PCR-RFLP typing method for the caprine Mhc classII DRB gene. Vet. Immunol. Immunop. 55, 255–260.

The authors would like to thank Professors MingJia Li, Ci Bian and Hong Wei for the collection of goat blood samples or providing the DNA samples, also thank the professors in Laboratory of Molecular Biology and

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