Identification of SNPs in the MAP4K4 Gene Coding Sequence and Their Association with Mastitis Susceptibility/Resistance in Nili Ravi Buffaloes

Background: The Nili-Ravi water buffalo (Bubalus bubalis) stands out as the premier domestic dairy animal, renowned for its exceptional milk production capabilities. Its significance in agriculture is underscored by its remarkable efficiency in converting crop by-products into valuable resources, coupled with its ability to thrive and maintain productivity amidst changing climatic conditions. An instrumental player in inflammation regulation, the Mitogen-Activated Protein Kinase 4 (MAP4K4) gene holds a pivotal role. The primary objective of the presented research communication was to investigate the potential correlation between specific single nucleotide polymorphisms (SNPs) found across the entirety of the MAP4K4 gene’s sequence and the susceptibility of Nili Ravi buffaloes to mastitis. Methods: A total of 40 whole blood samples were collected having mastitis positive (n=20) and an equal number (20) of healthy, normal counterparts. From total 40 samples, RNA extracted, cDNA was synthesized and the PCR products generated by employing specific primers were meticulously purified using a gel purification kit after agarose gel electrophoresis. Subsequently, the MAP4K4 gene was amplified and subjected to sequencing procedures. The chromatogram data obtained from sequencing were subjected to homology analysis using the Finch-TV tool. This scrutiny unveiled the presence of a total of seven single nucleotide polymorphisms (SNPs). To elucidate the potential connection between the identified SNPs and the animals’ susceptibility or resistance to mastitis, a chi-square test was employed. Result: In conclusion, Identified seven SNPs at locations 173, 226, 252, 329, 400, 410 and 459. In 3 SNPs, values were found to be less than 0.05 and in 4 SNPs values were found to be greater than 0.05. This confirmed the possibility of association of the MAP4K4 gene with the first 3 determined SNPs. The current genome association study showed the potential correlation between these significant polymorphisms and the incidence of mastitis in Nili Ravi buffaloes. As a result, the MAP4K4 gene may be an ideal candidate gene for dairy buffalo selection against mastitis and the discovered polymorphisms may one day serve as potential genetic markers.