Identification Of Elda-MHC Class â… , Class â…¡Genes And Assessment Of Adaptive Evolution Potential For The Pere David's Deer

Pere David's deer (Elaphurus davidianus) is a highly inbred species that arose from18founders but now comprises a population of about3000individuals. It is an endemic species of China but became extinct in China in the late19th century due to the war and flood. The current populations around the world all derived from18individuals that were housed in England's Woburn Abbey. In the1980s,77individuals were reintroduced from England to Beijing Nanhaizi Milu Park (38individuals) and Dafeng Milu National Nature Reserve (39individuals). Since then, the Pere David's deer population has expanded to a total of above1600individuals in China until2006. Thus, the Pere David's deer has recovered from the brink of extinction and become a successful example of how humans can conserve highly threatened species.The major histocompatibility complex (MHC) is a highly polymorphic multigene family critical to adaptive immunity and reproductive success. Thus it is a vital candidate gene family to examine individual and population viability. In this study we isolated MHC class â…  and class â…¡ genes and examined molecular variation using single strand conformation polymorphism-heteroduplex (SSCP-HD) and sequence analysis. The results are as follows:1) We developed a novel HURRAH method for isolating MHC loci, which includes the following steps:magnetic bead-based cDNA hybridization using biotinylated probes synthesized by PCR of universal primers; reconstitution of the SSCP-HD profiles of cDNA (r1) and genomic DNA (r2) to ensure that all of the relevant MHC sequences have been isolated; and analysis of heteroduplexes, which is used to estimate the total number of MHC class â…¡ loci.. This protocol is simple, efficient, reliable and could be applied to other mammals.2) Using this protocol we isolated ten class â…¡ sequences which were attributed to nine class â…¡ genes including DRA1, DRB1, DRB2, DRB3, DRB4, DQA1, DQA2, DQB1and DQB2. All of class â…¡ genes had normal start and stop codons, and the characteristics of classical class â…¡ genes. Thus the Pere David's deer possessed nine classical class â…¡ genes.3) Molecular examinations of Beijing and Dafeng populations revealed that all of nine class â…¡ genes were monomorphism at the peptide-binding region. Nine class â…¡ genes constituted two asymmetric functional MHC class â…¡ multi-locus haplotypes: DRA1*01-DRB1-DRB3-DQA1-DQB2(HI) and DRA1*02-DRB2-DRB4-DQA2-DQB1(H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class â…¡ haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes.4) Using this method we also obtained seven MHC class â…  genes, which we designated Fl, F12, G2,17, AF,18and C1. The Cl gene was an unexpressed pseudogene which we did not isolated its cDNA sequence. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and GC content revealed that F1, F12, G2and17were classical genes, AF was a nonclassical gene, and18and C1were pseudogenes.5) The molecular examinations showed distinct diversity patterns of class â…  genes from the common pattern. Most mammals have more polymorphic classical class â…  loci versus the nonclassical and neutral genes. In contrast, the Pere David's deer was found to be monomorphic at classical genes F1, F12, G2and17, dimorphic at the nonclassical AF gene, dimorphic at pseudogene18and tetramorphic at pseudogene C1. The adverse polymorphism patterns of class â…  genes might provide evidence for balancing selection to faster deplete MHC variation than drift in the bottlenecked populations.6) In total, we isolated16MHC genes of the Pere David's deer and obtained nine multi-locus MHC haplotypes. But only two multi-locus haplotypes were found from the perspective of classical antigen presentation fuction:DRA1-DRB1DRB3-DQA1-DQB2-F1-F12-G2-17(HI) and H2:DRA1-DRB2-DRB4-DQA2-DQB1-F1-F12-G2-17(H2). Consequently, we recommend that the scientific reproduction plans should be formulated, aiming at increasing the number of haplotype heterozygotes (H1/H2) in the descendants, in order to enhance the survival ability of this species.