| Objective:SNPs in MHC-DRB1exon2of Chinese Merino Sheep were analyzed through the experiment and then constructed its haplotypes. SNPs as well as haplotypes associated with Brucellosis susceptibility were preliminarily screened. The purpose of this study was to apply research experience about human diseases, and HLA haplotypes associations, to animal disease resistance genetics and breeding research, which provide the certain basis of the development of Brucella resistance molecular markers selection-assisted study in the future. This will be an important research direction to discover genetic susceptibility sites and will provide the basis for marker assisted selection for disease resistance.Methods:1. The Rose Bengal Plate Agglutination Test (RBPT) was used to detect positive Brucella antibodies in193Chinese Merino sheep. This research is based on the polymerase chain reaction(PCR) combined with SSCP technique to detect single nucleotide polymorphisms (SNPs) of MHC-DRB1exon2, and then cloned to allow sequencing of the different alleles. To determine the gene exon2polymorphism sites, molecular biology software was used for comparison analysis. For each SNP allelic and genotypic frequencies were compared between case and control samples by SHEsis online software, in addition it’s association with Brucellosis susceptibility was determined.2. PCR-direct sequencing method was used to detect the SNPs for40Chinese Merino sheep of the positive and negative Brucella in this study, then Hardy-Weinberg equilibrium and linkage disequilibrium were detected and analyzed. Haplotypes were established and their frequencies were analyzed by SHEsis online software. Initially speculated haplotype which may associated with Brucellosis susceptibility.Results:1. The Rose Bengal Plate Agglutination Test (RBPT) was used to detect the Chinese Merino sheep that expressed antibodies to Brucella. Out of193sheep tested,126were negative for Brucella, and67tested positive. The rate of positive Brucella was34.71%.2. Forty-one SNPs were found in MHC-DRB1gene exon2(270bp) from Chinese Merino sheep. Insertion and deletion site did not exist in these SNPs, and there were nine sites belonging to the PIP and thirty-two belonging to the SP. For MHC-DRB1exon2amino acid sequence were analyzed, and there were twenty-three sites belong to SAPs.3. The polymorphisms of HLA-DRB1exon2were analyzed,55SNPs and36SAPs were found. Compared with Chinese Merino sheep, only seven SNPs were same, and the others were different. Single amino acid polymorphism were more rich.4. The MHC-DRB1exon2sequences from different breeds of sheep were aligned. The results demonstrated that in different breeds of sheep, SNPs and SAPs were not the same in the270bp. 5. The distribution of C>T alleles at the109loci from the forty-one SNPs was significantly different between case and control samples(P<0.05). Association analysis was conducted for genotype of each SNP, showing that the distribution of genotype in case and control samples had no significant difference.6. According to conditions conforming to construct haplotype, there were29eligible SNPs sites were screened, and they were used to analyze LD and construct haplotypes. LD analysis found that MHC-DRB1exon2of Chinese Merino sheep had nine LD Block, and between each Block within SNPs two had strong LD, which was named Block1-Block9in turn. But we found that in different LD Block, the number of SNPs sites were different, Block7include A171G、G172C、A190G and G200T, Blockl、Block3and Block4only include two SNPs sites.7. By haplotype analysis, twenty-nine SNPs were used to construct haplotypes, but only nine haplotypes were analyzed. According to the haplotype frequency distribution difference in case and control samples, we found that Hap8and Hap9these two haplotypes frequency distribution difference were significantl(P<0.05).Conclusion;1. The MHC-DRB1gene exon2of SNPs in Chinese Merino sheep were analyzed and forty-one SNPs and twenty-three were found in270bp sequence. The results fully demonstrated that there were rich SNPs and SAPs existing in the sequence and showed rich polymorphism indeed.2. Because the sequence of MHC-DRB1exon2in sheep and human is highly homologous, and the HLA-DRB1exon2polymorphisms were analyzed. The results showed that SNPs and SAPs in human MHC-DRB1exon2were significantly more rich and complex than in Chinese Merino sheep.3. The MHC-DRB1exon2sequences from different breeds of sheep were aligned. The results demonstrated that in different breeds of sheep, SNPs and SAPs were not the same in the270bp.4. The association between polymorphism of MHC-DRB1genes exon2and Brucellosis susceptibility was analyzed, the results showed that MHC-DRB1exon2109C/T associated with Brucellosis susceptibility, and genotype of each SNP may did not associate with Brucellosis susceptibility. The results confirmed that DRB1gene indeed involved in antigen presented.5. Because LD existed, nine LD Block were found in MHC-DRB1exon2and strong LD between each two SNPs existed in every Block. The haplotype analysis found only nine haplotypes. According to the haplotype frequency distribution difference in case and control samples, we found that frequency of Hap8and Hap9distribution difference were significantly (P<0.05), initially speculated that these two haplotypes may associated with Brucellosis susceptibility. |
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