Characterization of major histocompatibility complex DRA and DRB genes of the forest musk deer (Moschus berezovskii)

The forest musk deer (Moschus berezovskii) is one of the most endangered species in China. Over the past decades, extensive hunting and poaching have pushed the forest musk deer to the edge of extinction, and conservation biologists are presently pursuing scientific management plans to rescue this species. The major histocompatibility complex (MHC), a cluster of genes responsible for antigen presentation, is a highly polymorphic genomic region in vertebrates that has become a popular functional marker system for studying adaptive variation. In this study, we developed locus-specific genotyping primers for exon 2 fragments of one DRA gene and one DRB locus of the forest musk deer using a suite of comprehensive methods that included universal primer amplification, genome walking, single-strand conformation polymorphism (SSCP), heteroduplex (HD) profiling, and sequence analysis. Each forest musk deer showed no more than two sequences per locus, confirming the specificity of our primers. Genotyping with these primers allowed us to identify two DRA alleles and six DRB alleles in a captive breeding population of the Sichuan Musk Deer Breeding Institution. For the DRA locus, we found a slightly higher observed heterozygosity (N _O=0.154) than expected (N _E =0.143). In contrast, the DRB locus showed a significant heterozygote deficiency (N _O=0.508; N _E=0.761; P<0.05), which was probably due to inbreeding in the captive population. An obvious excess of nonsynonymous substitutions over synonymous was observed at the antigen-binding positions of the DRA and DRB loci, showing the presence of positive selection in the forest musk deer DR genes. Finally, generation of phylogenetic trees for the DRA and DRB sequences of the forest musk deer and other ruminants revealed that the DRA and DRB loci identified in this study had homologous relationships with the known ruminant DRA and DRB genes. Based on this analysis, and to facilitate future studies, we named these novel loci Mobe-DRA and Mobe-DRB3.