ORIGINAL RESEARCH Sequence and topological characterization of Toll-like receptor 8 gene of Indian riverine buffalo (Bubalus bubalis) Praveen Kumar Dubey & Shubham Goyal & Jigyasa Aggarwal & Suresh Kumar Gahlawat & Periasamy Kathiravan & Bishnu Prasad Mishra & Ranjit Singh Kataria Accepted: 9 May 2012 / Published online: 24 May 2012 # Springer Science+Business Media B.V. 2012 Abstract In this study, buffalo (Bubalus bubalis) Toll-like receptor 8 (TLR8) gene has been characterized by sequence analysis and detecting polymorphism. Complete ORF of buffalo TLR8 gene was amplified using the RNA isolated from spleen tissue, which was found to be 3,102 nucleotides long encoding a 1,033 amino acid protein. Buffalo TLR8 had 10 nucleotide changes as compared to other livestock species resulting in six unique amino acid changes, four of them lying within leucine-rich repeat (LRR) domains. As compared to cattle (Bos indicus and Bos taurus), out of fifteen cysteine residues, fourteen were conserved and Cys at position 521 was replaced by Arg. Nine of the LRR domains had no amino acid change as compared to cattle, whereas LRR-C-terminus had maximum, five amino acid changes. Sequence characterization of 12 riverine and swamp buffaloes revealed presence of four polymorphic nucleotides, two of them were non-synonymous, one syn- onymous and one site in 3′UTR. PCR-RFLP genotyping of non-synonymous SNP 2758A>G (ILeu920Val) in Toll–in- terleukin-1 receptor domain of 463 swamp and riverine buffaloes showed a higher frequency of allele A in swamp (95 %) as compared to riverine (9.84 %) buffaloes. Keywords Bubalus bubalis . Toll-like receptor 8 . Sequencing . SNPs . Genotyping Introduction Toll-like receptors (TLRs) belong to a family of type I trans- membrane proteins, playing an important role in innate as well as adaptive immunity. Mainly expressed on cells of the immune system such as macrophages, dendritic cells, neu- trophils and monocytes, at least 13 members of the TLR family have been identified in mammals (Roach et al. 2005). Each member of the TLR family is capable of recognising a unique set of pathogen-associated molecular patterns, such as bacterial cell wall components which include lipopoly- saccharides, peptidoglycan, flagellin, single-stranded or double-stranded RNA genomes of viruses, fungal compo- nents, etc., and induce signals for the production of cyto- kines (Akira et al. 2006). Genes encoding 10 of these receptors have recently been mapped to the cattle (Bos taurus) genome (McGuire et al. 2006). Among the reported TLR family members, Toll-like receptor 8 (TLR8) recognises microbial single-stranded RNA and is localised in intracellular compartments such as endoplasmic reticulum (Zhu et al. 2009a). Activation of TLR8 induces production of proinflammatory cytokines like TNF-α and interleukin (IL)-12 and is a less efficient inducer of type I IFN (Gorden et al. 2005). The TLR7 and TLR8 genes lie adjacent to each other in the genomes of mammals and fish (Roach et al. 2005). Both genes are homologous to each other and are reported to be located on the X chromo- some in most of livestock species (Astakhova et al. 2009). In cattle, TLR8 consists of a single exon with a 3,102 nucleotide length coding 1,033 amino acids and 5′UTR and 3′UTR are yet to be reported (Cargill and Womack 2007). TLRs are composed of extracellular/ectodomains leucine-rich repeat (LRR) domains, which participate in pathogen recognition, and a cytoplasmic signalling domain P. K. Dubey : S. Goyal : J. Aggarwal : P. Kathiravan : B. P. Mishra : R. S. Kataria (*) National Bureau of Animal Genetic Resources, GT Road By-Pass, Karnal 132 001 Haryana, India e-mail: katariaranji@yahoo.co.in S. K. Gahlawat Department of Biotechnology, Ch. Devi Lal University, Sirsa, Haryana, India Trop Anim Health Prod (2013) 45:91–99 DOI 10.1007/s11250-012-0178-1