Genetic Variation and Evolutionary Conservation of Innate Immunity Genes in Pakistani Yak (Bos grunniens)

Toll-like receptor genes play a central role in innate immune recognition and inflammatory signaling but the coding-region variant and structural implication has not been well studied in yak (Bos grunniens), a species that are particularly vulnerable to high-altitude pressures and pathogens. The purpose of the study was to identify coding variants in innate immune genes (TLR2, TLR4 and SLC11A1) in Pakistani yak and to assess their evolutionary and structural value. TLR4gene fragments along with TLR2 and SLC11A1 were amplified and Sanger sequenced. Alignment of the sequences showed that there were 4 synonymous replacements in TLR4 exon 3 (c.813 G>A, c.966 T>C, c.1101 T>C, and c.1153 T>C), and no variation was observed in TLR2 and SLC11A1. Comparison of motifs in yak, cattle, sheep and goat revealed 10 highly conserved motifs with very high degree of enrichment implying a lot of evolutionary constraint. Phylogenetic reconstruction showed many of the clustering mediation of yak with cattle and general conservation of TLR4 in Bovidae. Combined synonymous substitutions were found to change the topology of mRNA folding, enhance the stability of the ensemble and significantly increase the diversity of the ensemble, indicating that enhanced conformational plasticity was achieved without generating new protein sequence through RNA secondary structure analysis and thermodynamic ensemble analysis. These results show that TLR4 in yak is subjected to intense purifying selection on the protein scale, whereas synonymous variation might mediate the structure dynamics in the transcript scale, implying that an immune gene evolution process has its potential regulatory aspect.