Association Analysis of MBL1 Gene SNPs, Genotype and Haplotypes with Clinical Mastitis in Murrah Buffaloes

Mannose-binding lectins (MBL1) gene a most important constituent of immune response system of an organism, the primary role of this gene in the classical and lectin-activation pathways is to provide protection against various dieases or bacterial pathogens. In this study, eight novel SNPs identified in the promoter region of MBL1 gene were 154C>T, 235G>A, 252A>T, 265C>A, 268T>C, 282G>A, 431G>A and 551C>G, when compared to reference sequence of Bubalus bubalis (NCBI accession number KC415281). Genomic DNA isolated from 200 lactating Murrah buffaloes was amplified for the targeted sequence of MBL1. PCR products were custom sequenced, edited and used for further analysis. The results indicated that only 551C>G polymorphism locus met Hardy–Weinberg equilibrium (c2 (2df) = 4.4: P=0.11). Pair linkage disequilibrium analysis and haplotype construction of MBL1 gene were performed using SHEsis software. Pair linkage disequilibrium analysis revealed moderate to strong linkage disequilibrium between the eight SNPs loci A total of 17 haplotypes were generated from eight SNPs in promoter region of MBL1 gene. Association between eight SNPs of MBL1 gene in Murrah buffalo has been analyzed and relative risks of the alleles for clinical mastitis were estimated with an odds ratio. Allelic association analysis showed that T allele of 154C>T, A allele of 235G>A, A allele of 252A>T, A allele of 265C>A , T allele of 268T>C, A allele of 282G>A , A allele of 431G>A and G allele of 551C>G had significant association with increased risk of clinical mastitis (P< 0.01). Haplotype analysis showed that Hap15 (CAAATAAG) and Hap16 (TAAATAAG) i.e. the haplotypes containing all 8 “at-risk” alleles, were significantly associated with clinical mastitis (P< 0.01). Hap6 (CGAATAAC) and Hap8 (CGTCCGGC) were significantly associated with a lower risk of clinical mastitis in Murrah buffaloes (P< 0.01).On the other hand, the Hap6 (CGAATAAC) and Hap8 (CGTCCGGC) which are unrepresented in affected group and without “at-risk” alleles were significantly associated with a lower risk for clinical mastitis (P<0.01).These findings indicated a MBL1 gene polymorphism could be used as genetic marker for mastitis resistance in Murrah buffaloes.