Polymorphisms And Haplotypes Of The OLA-DQB1 Gene Exon2 Of Chinese Merino Sheep And Association With Brucellosis Susceptibility

Objective:The SNPs of OLA-DQB1 gene exon2 in different case and control Chinese Merino sheep were detected by the experiment, and Haploview4.2 was used to construct its haplotypes to analyze the association between the SNPs loci and Brucellosis susceptibility as well as the haplotypes with its susceptibility. By referencing the research experience of HLA-DQB1 gene which associating to disease, using SNPs of OLA-DQB1 gene to do correlation analysis with sheep diseases, thus will be a referable direction to carry out resistance marker assisted selection research of Brucellosis, to find the susceptible genetic loci will accelerate the process of molecular genetic disease resistance breeding.Methods:1. Using The Rose Bengal Plate Agglutination Test(RBPT) to detect positive and negative Brucella antibodies in 408 Chinese Merino sheep, and then the SNPs loci were detectd by PCR-SSCP technique from OLA-DQB1 gene exon2, selecting different alleles to complete clone sequencing. The sequencing results were compared by Molecular Biology software, and to contrast its SNPs and SAPs with humans’. By using Pop Gene1.32 to finish each SNP’s gene frequency and genotype frequency and find the special SNPs which associated with Brucellosis susceptibility.2. Selcting 44 positive and negative individuals to use PCR product direct sequencing method to detect the OLA-DQB1 gene exon2 and analyze whether its Hardy-Weinberg could achieve balance. After that, the screened SNPs were used for analyzing its linkage disequilibrium by Haploview4.2 and PHASE2.1. The eligible SNPs were using for constructing haploid linkage map. Finally, the PHASE2.1 will speculate out the relevant haplotype which associated with Brucellosis susceptibility.Results:1. 76 positive and 332 negative Chinese Merino sheep were detected by using RBPT, Brucella positive detection rate was 18.63%.2. 43 SNPs were detected through the OLA-DQB1 gene exon2(270bp) of Chinese Merino sheep in this study, no insertions and deletions loci in all the SNPs. These loci include 41 parsimony informative sites and 2 uninformative sites, the single variable sites were 35. The translated amino acid sequence(89aa) were analyzed and 29 amino acid polymorphic sites were found that belonging to sense mutation.3. 56 SNPs and 34 SAPs were found in different HLA-DQB1 gene exon2. By comparing with variation loci of Chinese Merino sheep, humans had more abundant SNPs and SAPs than sheep. Among these variation loci, 7 SNPs loci and base were definitely uniform.4. The allele of 196G/A existed highly significant difference(p<0.01) between case and control group among the 43 SNPs, and 211C/T similarly presented significant difference(p<0.05). What’s more, the genotype frequency of 196G/A still showed significant difference in both case and control group(p<0.05).5. By setting up conditions of constructing haplotypes, 20 SNPs were screened out from the detected polymorphism loci by software. LD analysis results showed that the Chinese Merino sheep existed 6 LD Block, and each Block had strong LD, named Block1-Block6. More importantly, each LD Block described different SNPs from the Linkage disequilibrium map.6. 6 haplotypes were inferred by 20 eligible SNPs from Chinese Merino sheep and Hap5 showed significant difference between case and control group, this haplotype test were below the 0.05 level of significances.Conclusion:1. By detecting the polymorphic sites in OLA-DQB1 gene exon2 of Chinese Merino sheep, 41 parsimony informative sites and 29 amino acid polymorphic sites were found in all the 270 bp. The results illustrated that the number of SNPs and SAPs in this sequence were so rich and this phenomenon was consistent with the diversity of immune genetic coding products.2. Because sheep existed high level of homology with humans, to analyze and compare the polymorphisms of HLA-DQB1 gene with sheep, the result showed that the SNPs and SAPs of humans were more abundant than sheep.3. The data indicated that 196G/A and 211C/T existed highly significant and significant difference in both case and control group after doing correlation analysis with these SNPs which were detected from OLA-DQB1 gene exon2. Besides, the genotype frequency of 196 site was also significant difference in both of them. To speculate that they might be associated with brucella susceptibility, reflected that DQB1 gene might be involved in the different types of antigen presentation activities.4. According to the phenomenon that adjacent SNPs might exist linkage disequilibrium, contacting all the eligible SNPs with brucella susceptibility, 6 LD Block were found and the SNPs were strong linkage disequilibrium. 20 SNPs were futher screened to construct 6 haplotypes which Hap5 test were below the 0.05 level of significances, thus conclusion could be drown that this haplotype closely related to brucella susceptibility of Chinese Merino sheep.