Polymorphism And Maintaining Mechanism Of Mhc-DRB Gene Of Two Species In Muntiacus

Muntjacs are famous for their chromosomes constitute and rapid and radical karyotypic diversification compared to other mammals,constituting a paradigm for the studies of karyotypic evolution. The major histocompatibility complex (MHC), unique to vertebrate, plays important roles in immune response and mate choice. Polymorphisms of the MHC genes are maintained by some forms of balance selection. Therefore, the genes in the MHC are thought to be of important adaptive significance and are considered to be the best candidate markers in the research of adaptive evolution. To now, no information related to MHC genes of muntjacs was reported though some mhc genes of other deer have been analyzed. Of Muntiacus, Black muntjac (Muntiacus crinifrons) and Chinese muntjac are two sympatric species in southern Anhui province. However, the two species have distinct population history, Karyotypic characterize, geographic distribution and current population Demography. All these differences between the two animals provide us a good opportunity to insight into the polymorphisms and maintaining mechanism of MHC-DRB gene of muntjacs. These results may provide new evidences for clarify the evolutionary mechanism of DRB gene of Cervidae. Furthermore, the results will probably contribute to establish reasonable protection strategy for the endanged black muntjac. The main results were as follows.1. A pair of primers (LA31 and LA32) specific to Bola-DRB3 was used to amplify genomic DNA of the Black Muntjac (Muntiacus crinifrons) and Chinese muntjac (Muntiacus reevesi). Based on polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), and sequencing technologies, eleven and twenty distinct DRB exon 2 alleles were respectively obtained from 44 Black Muntjac and 12 Chinese Muntjac. Taking into account the number of alleles and nanalyzed samples, the Chinese muntjac may possess higher allele diversity than that of black muntjac. Two DRB loci for Chinese muntjac could be determined because more than two alleles were identified from six of 12 Chinese muntjac individuals. However, at least three loci could be determined for black muntjac as five alleles were identified from 3 Black Muntjac individuals (samples 3, 23 and 34).2. Based on multiple sequence comparison, sequence of muntjacs DRB alleles showed high similarity to that of the expressed Bola-DRB3 locus. No insertion, deletion or stop codons within the nucleotide sequences were detected. Furthermore, the amino acid mutation sites were mainly confined in the putative peptide binding region (PBR). These results implied that the loci isolated in this study are probably expressed and functional. Frequency of nonsynonymous (dN) is greater than that of synonymous (dS) (p<0.05) in PBR of both species suggesting that DRB genes of the two species might had historically undergone positive selection. Which was further confirmed by the results of likelihood ratio test (LRT) from model M7 and M8 of program CODEML. In total, six and five robust positive selection amino acid sites, of which, 3 and 5 sites located in PBR, were detected by a Bayesian method for Chinese muntjac and black muntjac, respectively. These results provide evidences that positive selection is one of the main mechanisms in maintaining DRB gene polymorphism of the two species.3. A neighbor-join (NJ) tree was constructed based on DRB gene exon 2 sequences of several deer including all sequences obtained in the present study. In the NJ tree, DRB alleles of the two muntjacs showed a mosaic pattern with that of other deer, implying a trans-species evolution pattern in maintaning DRB allele polymorphism. Moreover, one DRB allele of black muntjac (MucrDRB8), two alleles of Chinese muntjac (MureDRB1 and MureDRB11) and two DRB alleles of red deer (CeelDRB34 and CeelDRB46) were closely clustered with DRB3 alleles of cattle, suggesting some DRB alleles of these deer might have an ancient lineage.