Development Of The MHC Class Ⅰ Molecular Marker System And Adaption Evolution Analysis For The Giant Panda

The giant panda(Ailuropoda melanoleuca) is an endangered species endemic to China with state grade Ⅰ class protection. Unfortunately, due to serious habit fragmentation, the wild populations were distributed in six completely isolated mountain ranges of China:Qinling, Minshan, Qionglai, Daxiangling, Xiaoxiangling and Liangshan.Hundreds of species went extinct due to climate change in the late Pleistocene. However, the giant panda survived to become a living fossil, making it interesting to investigate the adaptive molecular evolutional mechanism of this old species.The major histocompatibility complex (MHC) is one of the most polymorphic functional genes in the genome of vertebrates. It plays an important role in maintaining animals adapt to the changing environment due to its ability to encoding molecules those present pathogen peptides to immune cells and trigger an immune response. It forms a molecular marker system to examine adaptive evolution of veterbrates. Here, we constructed BAC-based contig maps containing the MHC class Ⅰ genes and then sequenced these contigs to isolate all the MHC class Ⅰ genes. We developed locus-specific genotyping and haplotyping techniques to form a good molecular marker system of MHC class Ⅰ genes. We investigated the genetic variability and analyzed mountain differentiation and the maintaining mechanism of MHC diversity in six wild populations for the giant panda. The main results with neutral microsatellites data are as follows:(1) We isolated16positive BACs and they were assembled into two contigs, one spanning380kb and one spanning85kb. GENESCAN predicted that there were six MHC class Ⅰ genes in these two contigs, and we designated them Aime-C, Aime-F, Aime-I, Aime-K, Aime-L and Aime-1906, according to their contig locations.(2) Analyses of class Ⅰ gene tissue expression patterns and full-length cDNA sequences revealed that the Aime-C,-F,-I and-L genes could be considered classical class I loci, due to their extensive expression patterns and normal exonic structures; in contrast, Aime-K and-1906appeared to be nonclassical genes based on their tissue-specific expression and the presence of an abnormal exon7in both genes.(3) We established locus-specific techniques for genotyping exons2and3and haplotyping long genomic fragments spanning exons2and3for the four classical MHC class I genes (Aime-C,-F,-I and-L) and then we validated the reliability of our genotyping and haplotyping techniques in captive giant pandas for whom we had full pedigree information.(4) We investigated genetic variations for218individuals from six wild populations and found that Aime-F was monomorphic, whereas Aime-C,-I and-L were polymorphic. We identified23linked haplotypes across the three polymorphic loci:Aime-C (9), Aime-I (7) and Aime-L (7). Compared to other endangered carnivores, the giant panda exhibited a moderate level of genetic diversity of the MHC class I genes.(5) Balancing selection on MHC class I genes of the giant panda was supported by several lines of evidence1) all three polymorphic MHC class I genes of the giant panda (Aime-C,-I and-L) revealed an excess of nonsynonymous over synonymous substitutions at their ABS sites;2) the lower differentiation was observed among the MHC I genes compared to the microsatellites;3) sharing of many alleles across divergent mountains in the giant panda.(6) Combined with the neural microsatellites data in this study, we revealed that the Qinling population showed the most significant genetic divergence with other five populations (P(MHC)=0.004; P(microsatellites)=0.010). This result supports the conclusion of Qinling population as a separate subspecies from the adaptive evolutional perspective. As to Sichuan subspeicies, Minshan and Qionglai populations formed a compound genetic unit-"Minshan-Qionglai", whereas the other three populations formed another genetic unit-"Daxiangling-Xiaoxiangling-Liangshan". On basis of our findings, we suggested three separate management units should be defined for the protection of the giant panda:"Qinling","Minshan-Qionglai" and"Daxiangling-Xiaoxiangling-Liangshan".