Isolation And Identification Of SSR And MHC ClassⅡGenes And Analysis Of Population Genetic Structure For The Golden Pheasant

Golden pheasant(Chrysolophus pictus), belong to Chrysolophus, Phasianidae, Galliformes. It is endemic to China and is a national second-class protected species. The habitat of this species has fragmented over years, which is caused by habitat destruction and excessive hunting. In this research, we isolated SSR loci and MHC loci, and set up two sets of molecular markers. Using this system to 13 populations, we wanted to explain the impact of habitat fragmentation on population genetic structure, and to explore the environment adaptability when the species in the stress of fragments.Firstly, we isolated fourteen SSR loci using magnetic bead hybridization method, and established a set of species-specific SSR primers in golden pheasant. Furthermore, 191 alleles were found in thirteen populations. The observed heterozygosity was ranged from 0.358 to 0.930. According to the results of STRUCTURE, thirteen populations were divided into four regions{SC (JG, QC, LC), Central (QJ, HN, GZ), QL (FP, SYG), North (LCP, NX, NS, ZZ, TS)} (grouping A). The grouping was proved by AMOVA with significant genetic variation (FCT>FSC, P<0.001) between four regions. We also constructed phylogenetic trees using Nei's distance and Chord distance, and these were corresponding to the results of STRUCTURE.Meanwhile, we constructed a new method named "Reverse-4D", which was suitable for birds. The genome of bird is not so large, so it is easier to target genes in a BAC library. This new method was convenience and economical, and the time need for constructing a BAC library was only 1/10 of traditional 4D approach. When targeting DNA fragment of 100-160kb, the amount of work was only 1% of the traditional way. So this modification in reverse-4D method greatly accelerated the progress of isolating few large genomic fragments or BACs.We successfully isolated 7 cloned with MHC using reverse-4D BAC library in golden pheasant. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes:partial BG-like, Blecl, two NKs, three MHC classâ…¡B loci, TAPBP, BRD2, DMA, two DMBs, two MHC class I loci, TAP1, TAP2, C4, CenpA, CYP21 and partial TNXB. Chpi-MHC showed good synteny with that of chicken. The notable differences between these two species were the numbers of classâ…¡B loci and NK genes and the inversions of TAPBP gene and TAP 1-TAP2 region.The set of locus-specific primers ofâ…¡B were constructed based on the full sequences of MHC genes in this species. The PCR-SSCP was used to compare variation by locus inâ…¡B among individuals. We isolated 18,7 and 7 alleles ofâ…¡B 1,â…¡B2 andâ…¡B3 respectively. The analyze show positive selection, co-evolution and tran-species evolution in MHC of golden pheasant. However, the analysis of BLA indicated it was monomorphic in this species. The investigation of IIB show frequencies was significant different between populations. Allelesâ…¡B1*01,â…¡B2*01 andâ…¡B3*02 were dominant in three loci respectively. According to STRUCTURE, the thirteen populations were divided into two regions{(JG, QC, FP, SYG, LCP, NS, NX, ZZ, TS) (LC, QJ, HN, GZ)}(grouping B). And grouping B was also verified in MHC by AMOVA (FCT>FSC, P<0.001).The results of STRUCTURE were different in SSR and MHCâ…¡B. LC was gathered with HN, QJ and GZ populations in grouping B. It might be due to the selection forced by environment. The other nine populations which all located on the north side of Changjiang River, were gathered into one cluster in grouping B. Homogenization might be the reason for this phenomenon. So, it was supposed that Changjiang River had effect on environment which effecting selection of MHC genes. We also used G'st to compare the genetic differentiation of MHC and SSR. Lower values were found in MHC, which was also beyond the 95% CI of SSR. The mean value of G'st-SSR/G'st-MHC was 2.036, which indicated balancing selection played an important role in the evolution of MHC in golden pheasantIn sum, our present results suggest some proposals for this species. Firstly, according to MHC the populations were divided into two regions, which were separated by Changjiang River. Therefore, the introduction of populations between the south and north side of Changjiang River can be helpful for the improvement of genetic diversity. Secondly, there were unique pattern of MHC in FP and LC, and it would be an important resource for the relationship between MHC haplotypes and pathogens.