Isolation Of MHC Class Ⅰ Loci And The Adative Evolutionary Studies Of MHC For The Golden Pheasant

Golden pheasant(Chysolophus pictus) is a near threatened species endemic to China with national second-class protection. In rencent years, there is a sharp decline in the number of golden pheasants, which is caused by severe fragmentation of habitat and excessive hunting. Hence, it is necessary to investigate the genetic diversity and explore the adaptive evolution mechanism of this species under the stress of habitat fragmentation.The major histocompatibility complex (MHC) is one of the most important gene family in the vertebrate immune system, encoding diverse glycoprotein molecules, which responsible for binding and presenting pathogen peptides to T cells in order to trigger an appropriate immune response. The diversity of MHC genes may directly influence the survival of individuals against pathogen disease and it also plays a key role in the adaptation of species to the changing environment. It becomes a popular molecular marker system in the adaptive evolutionary study of vertebrates. In this study, we isolated all the MHC I gene sequences in the golden pheasant, then developed locus-specific genotying primers and finally set up a good molecular marker system of classical MHC I loci for this endangerd bird. We investigated the genetic variability, analysed the maintenance mechanisms of MHC polymorphism, and discussed population structuring in twelve wild golden pheasant populations. The main results and conclusions are listed below.(1) We isolated all the MHC I genes and got six full-length genomic sequences from the same individual and designated them A-F according to an alphabetical order. The results of cross-locus amplication showed A/C and B/D belonged to Chpi-IA1and Chpi-IA2respectively.(2) We isolated a positve BAC clone C11, which is about80kb in length, and forms a contig with the previously published S2BAC clone (96kb). There were three MHC I genes inclued in this contig:the former two genes Chpi-IA1and Chpi-IA2, and the new identified gene Chpi-IA3. The novel MHC Ⅰ gene was comprised of sequences E and F and was only found in C11.(3) Analysis of tissue expression revealed that IA1and IA2could be regarded as classical loci due to their extensive tissue expression while IA3appeared to be nonclassical one without expression patterns, though the predicted cDNA full-length sequences had normal exonic structures and typical characteristics of classical class MHC I genes.(4) Combining the PCR-SSCP and sequencing methods, we succeeded in establishment of locus-specific genotyping techniques for exon2and exon3for the two classical MHC class I genes (Chpi-IA1and Chpi-IA2). Then we investigated genetic polymorphisms of334individuals from12wild population of the golden pheasant and identified41exon-2alleles and39exon-3ones across the two polymorphic MHC class I genes:Chpi-IA1-E2(14), Chpi-IA1-E2(11), Chpi-IA2-E2(28) and Chpi-IA2-E3(27).(5) We found evidences of balancing selection in the MHC class I genes of the golden pheasant; showing much higher amino acid substibutions in the PBR than the non-PBR, having an exccess of nonsynonymous nucleotide substitution over synonymous nucleotide substitution at the PBR sites of al domain in both polymorphic MHC class I genes, presenting eight out of ten positively selective sites in the PBR, observing sharing of alleles in high frequencies over all the populations.(6) Recombination is also an important evolutionary mechanism of MHC class I genes in the golden pheasant. The phylogenetic tree showed that about37.0%Chpi-IA2-E2alleles and all Chpi-IA1-E2alleles were grouped together. The three methods (MaxChi2,3Seq and GARD), all verificated that the inter-gene recombination existed between Chpi-IA2-E2and Chpi-IA1-E2, and found that those Chpi-IA2-E2alleles might be recombinants.(7) Class I gene-based differentiation coefficients revealed there were no significant genetic divergence between the populations of the south and those of the north of Qinling Mountain, while significant differentiation between populations of south to the Yangtze River and all populations of north to the Yangtze River. Combined with the AMOVA and Bayesian clustering results, it supported twelve populations could be divided into two regions,{(LC, HN, QJ, GZ)(LX, TS, BJ, FP, LN, JQ)}.There were much genetic divergence between the two geographic regions due to the seperation of Yangtze River.