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

Abstract

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.

Identification of single nucleotide polymorphisms in Toll-like receptor candidate genes associated with tuberculosis infection in water buffalo (Bubalus bubalis)

“…They are characterized by an extracellular N-terminal domain constituted by 16 to 28 leucine rich repeats (LRR) involved in ligand recognition [ 16 ], and an intracellular C-terminal domain known as the toll/IL-1 receptor (TIR) domain, required for the interaction and recruitment of various adaptor molecules to activate the downstream signalling pathways [ 17 ]. In water buffalo, each cell contains 25 chromosome pairs ( Bubalus bubalis 2n = 50) [ 18 ], and the TLRs 2 and 9 loci are localized on chromosomes 17 and 21, respectively, while the TLR 4 locus is located on chromosome 3 [ 19 ].…”

Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

“…The frequency of AA genotype was significantly higher (p≤0.05) in healthy animals and indicated the association of AA genotype with resistance to mastitis. Mitra [ 22 ] studied TLR-4 gene of Murrah buffaloes and was found 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 showed twelve SNPs with six non-synonymous SNPs resulting in a change in amino acids.…”

Association of <i>TLR4</i> and <i>CARD15/NOD2</i> polymorphisms with SCC in Holstein–Friesian cattle

“…The bovine TLR4 gene coding region is 2526 bp long, consists of 3 exons and is located on chromosome BTA 8. Exon 1 includes coding base pairs 1-95, exon 2 consists of base pairs 96-260 and exon 3 comprises base pairs 261-2526 (Mitra et al, 2012). More than 40 SNPs (single nucleotide polymorphisms) of the bovine TLR4 gene have been found to date, which means an average of 1 SNP per 90 bp (Wang et al, 2008).…”