Polymorphisms And Haplotype Recombination Of The DQA1, DQA2 And DRB3 Gene In Tibetan Sheep

With the view of studying polymorphism of DQA1,DQA2 and DRB3 genes exon 2 in Tibetan sheep, determining the number of alleles, nucleotide polymorphism sites, amino acid polymorphism sites, haplotype recombination of alleles, genetic relationship among every allele and evolutionary significance, as well as the possibility of DQA1,DQA2 and DRB3 genes as the molecular genetic markers. Using PCR-SSCP method, we detected 900 Tibetan sheep DQA1,DQA2 and DRB3 gene exon 2 alleles at the first time, and cloning and sequencing every allele which comes from variation in population, then analyzing the haplotype sequence data of allele. We got the following conclusions:1.By PCR-SSCP detecting, cloning, sequencing and analysising of sequence comparison on DQA1,DQA2 and DRB3 gene in 900 Tibetan sheep,17 DQA1 alleles, 16 DQA2 alleles and 51 DRB3 alleles were obtained. There are 5 new DQA1 alleles,8 new DQA2 alleles and 50 new DRB3 alleles in them were found first time in this study. The appearance of new DQA1,new DQA2 and new DRB3 alleles in Tibetan sheep may be related to the difference of geographical surrounding and climatic conditions between the Tibetan sheep and exotic sheep breeds.2.There are 218 individuals (24.22%) that hadn't detected DQA1 gene in 900 Tibetan sheep, belong to the DQA1 null haplotype. There are 56 nucleotide polymorphism sites were found in 16 DQA1 gene exon 2 alleles,71 nucleotide polymorphism sites in 16 DQA2 gene exon 2 alleles, and 91 nucleotide polymorphism sites in 51 DRB3 gene exon 2 alleles. The results showed that DQA1,DQA2 and DQA3 genes had high polymorphism, which probably attribute to the consistency of Tibetan sheep trait by living in wicked environment, high adaptability, extensive resistance, and high disease resistance. DRB3 gene has the richest polymorphism in the 3 MHC genes,DQA2 and DQA1 take the second place.3.There are 27 amino acid polymorphism sites were found in 16 DQA1 gene exon 2 alleles,40 amino acid polymorphism sites in 16 DQA2 gene exon 2 alleles, and 50 amino acid polymorphism sites in 51 DRB3 genes exon 2 alleles in Tibetan sheep.4.Phylogenetic analysis of the DQA1 and DQA2 gene exon 2 haplotypes in Tibetan sheep showed that two different clusters were found, which indicated that DQA1 and DQA2 gene may be developed into two main type alleles by 2 initially alleles.However, the DRB3 gene has 3 clusters, it showed that DRB3 gene were delveloped into three main type alleles from 3 initially alleles, or came from the first branch and second branch's evolution or mutation.5.In 16 DQA2 alleles,*N,*K and *F alleles belong to the DQA2-like gene. There are not detected DQA1 genes in the individuals with DQA2-like gene. It showed that DQA2-like gene probable took up the locus of DQA1 gene when DQA1 gene was deletion, and DQA2 gene had duplication in MHC genes as well.6. DQA1,DQA2 and DRB3 genes in Tibetan sheep and cattle has high homology, it indicated that sheep and cattle DQA1,DQA2 and DRB3 gene may be derived from primordial sequences that were present in a common ancestor and have persisted in the sheep and cattle populations since their divergence, and the observation that sheep and cattle share similar allelic sequences may be evidence of the need for a specific immune response to a common pathogen.7.Thirty five types of "DQA1-DQA2" gene haplotypes, seventy eight types of "DQA2-DRB3" haplotypes and one hundred and five types of "DQA1-DQA2-DRB3" haplotypes were found in this study. The number of haplotypes is less than theoretical value, and haplotype frequency distribution is extremely imbalance, showed that the DQA1,DQA2 and DRB3 genes haplotypes have linkage disequilibrium phenomenon. It was the first time found that haplotype recombination of DQA1 and DQA2 genes in MHC-DQ subregion with DRB3 gene in MHC-DR subregion8.DQA1, DQA2 and DRB3 genes can be used as molecular genetic markers in sheep, during the selection of disease resistance related to DQA1,DQA2 or DRB3 gene, combined with "DQA1-DQA2-DRB3" haplotype choice, will enhance the selection reliability.