Nucleotide Sequencing and SNP Detection of Toll-Like Receptor-4 Gene in Murrah Buffalo (Bubalus bubalis)

Toll-like receptor-4 (TLR-4) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections. The present investigation was carried out to study nucleotide sequencing and SNP detection by PCR-RFLP analysis of the TLR-4 gene in Murrah buffalo. Genomic DNA was isolated from 102 lactating Murrah buffalo from NDRI herd. The amplified PCR fragments of TLR-4 comprised of exon 1, exon 2, exon 3.1, and exon 3.2 were examined to RFLP. PCR products were obtained with sizes of 165, 300, 478, and 409？bp. TLR-4 gene of investigated Murrah buffaloes was highly polymorphic with AA, AB, and BB genotypes as revealed by PCR-RFLP analysis using Dra I, Hae III, and Hinf I REs. Nucleotide sequencing of the amplified fragment of TLR-4 gene of Murrah buffalo was done. Twelve SNPs were identified. Six SNPs were nonsynonymous resulting in change in amino acids. Murrah is an indigenous Buffalo breed and the presence of the nonsynonymous SNP is indicative of its unique genomic architecture. Sequence alignment and homology across species using BLAST analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with Bos taurus, Bos indicus, Ovis aries, Capra hircus, and Homo sapiens, respectively. 1. Introduction India is of a fortune position of having the world’s best breeds of buffaloes for milk production. Special attention has to be focused on Murrah breed of Buffalo whose breed average milk production is about 2200？kg per lactation. Buffalo contribute more than fifty percent milk to the total milk produced in India. However, due to increased prevalence of infections, the realization of their true genetic merit has been hampered. Among infectious diseases, mastitis, an inflammatory disease of the mammary gland generally caused by intramammary infections, is the most common, costly, and devastating disease in dairy animals. Therefore, attention needs to be focused to study the genes involved in disease resistance, especially for mastitis. Genes associated with immune responses of the mammary gland are potential markers because of their importance in mastitis. The toll-like receptor-4 (TLR-4) is an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections [1, 2]. Its role in pathogen recognition and subsequent initiation of the inflammatory and immune responses, and highly polymorphic nature in the bovine species, make it a suitable candidate gene for use in marker-assisted selection for enhancing disease resistance in dairy animals [3]. The TLR-4 gene coding