【作者】 何珂；

【导师】 方盛国； 万秋红；

【作者基本信息】 浙江大学 ， 动物学， 2012， 博士

【摘要】 红腹锦鸡(Chrysolophus pictus)隶属鸡形目(Galliforme)雉科(Phasianidae)锦鸡属(Chrysolophus),是我国特有的国家Ⅱ级濒危野生动物。多年来,生境破坏和过度猎杀,导致红腹锦鸡的栖息地严重片段化。本论文以生境片段化为切入点,并在分离SSR和Ⅱ类MHC位点以及建立两套分子标记系统的基础上,通过群体检测,探索生境片段化对种群遗传结构的影响,以及种群在片段化胁迫下的环境适应性等科学问题。首先,利用磁珠富集方法共分离获得了14个红腹锦鸡特异的多态性SSR位点,并以此建立了物种的SSR分子标记系统。进一步地,运用SSR对红腹锦鸡野外现有的13个片段化种群进行群体检测,得到191个等位基因,其观察杂合度在0.358到0.930之间。通过STRUCTURE软件分析,发现13个种群被分为四大区域{四川(剑阁、青川、利川)；中部(黔江、湖南、贵州)；秦岭(佛坪、石印沟)；北部(龙草坪、宁陕、临夏、宝鸡、天水)｝(分组A)。通过AMOVA验证了这四大区域之间存在显著的遗传变异(FCT>FSC,P<0.001)。利用SSR数据生成的Nei氏标准遗传距离和余弦距离构建了种群地理系统树,该结果证明各地理种群之间的关系和STRUCTURE分析结果非常吻合。与此同时,以红腹锦鸡为对象,首次建立了适于鸟类基因组BAC文库构建的"Reverse-4D"新方法。‘’Reverse-4D"方法简便,省时且节俭经费,用之构建一个鸟类的基因组BAC文库,仅需传统“4D”方法的1/10左右的时间。特别地,筛选一个100-160kb的大片段DNA,则仅需传统BAC文库筛选方法的1%左右的工作量,达到了快速、高效和便捷的目的。运用"Reverse-4D"方法在成功构建红腹锦鸡基因组的"Reverse-4D" BAC文库的基础上,筛选得到7个MHC质粒。在S2质粒中包含20个基因,部分的BG基因、3个Blec基因、3个ⅡB基因、TAPBP基因、BRD2基因、DMA基因、2个DMB基因、2个MHC IA基因、TAP1基因、TAP2基因、C4基因、CenpA基因、CYP21基因和部分的TNXB基因。通过Chpi-MHC分析,发现红腹锦鸡和原鸡之间的基因相似度和基因排布顺序非常的相近,除了两个特殊的地方外,基本上呈现线性的同源性。主要的差异表现在红腹锦鸡有3个MHCⅡB基因,3个Blec基因,而原鸡只有2个MHCⅡB基因及2个Blec基因,说明在MHC家族中存在基因重复事件。另外在TAPBP基因和TAP1-TAP2区域,发生了基因的翻转过程。在红腹锦鸡MHC基因结构的基础上,随后成功建立起物种的MHCⅡB基因位点特异性扩增系统。通过PCR-SSCP在三个位点中分别得到18个、7个和7个等位基因,分析表明在ⅡB基因中存在正向选择、协同进化和跨物种进化现象。通过参照相近物种设计引物扩增ⅡA部分,种群研究调查显示其为单态。进一步地,通过MHCⅡB群体调查研究,发现不同种群之间等位基因频率存在很大的差别；其中ⅡB1*01、ⅡB2*01和ⅡB3*02分别是三个位点中的出现频率最高的等位基因。利用STRUCTURE软件对MHC数据进行分析,发现13个种群被分为两大区域{(剑阁、青川、佛坪、石印沟、龙草坪、宁陕、临夏、宝鸡、天水),(利川、黔江、湖南、贵州)}(分组B)；并且通过AMOVA验证了这两大区域之间存在显著的遗传变异(FCT>FSC,P<0.001)。通过SSR和MHCⅡB两种分子标记相比较,得到SRTUCTURE分组情况不同的现象,其中利川种群可能由于环境选择的压力,和湖南、黔江、贵州种群区域较为接近；而分组A中的另外9个种群在分组B中则聚集到一簇(均位于长江以北),说明这些种群之间存在同质化。因此认为长江这一地理结构对该物种的MHC基因进化存在一定的影响。通过G’st来比较MHC和SSR的遗传分化,发现G’st-MHC低于G’st-SSR,并且位于其95%置信区间之外G’st-SSR/G’st-MHC平均值的比值为2.036,说明在该物种中平衡选择对于MHC基因家族的选择起了重要的作用。综上所述,对于保护的建议措施如下：按照MHC的分析结果将其分布划分为两个大区域,在地理结构上为长江所间隔,因此对于江北和江南地区的种群可以适当进行种群引进,提高物种的遗传多样性；佛坪和利川种群具有较为独特的MHC结构,在该物种的抗病研究上是一个很好的试验对象,后期可以进行关于病原体如何导致MHC选择的相关研究。更多还原

【Abstract】 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 classⅡB 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 classⅡB loci and NK genes and the inversions of TAPBP gene and TAP 1-TAP2 region.The set of locus-specific primers ofⅡB were constructed based on the full sequences of MHC genes in this species. The PCR-SSCP was used to compare variation by locus inⅡB among individuals. We isolated 18,7 and 7 alleles ofⅡB 1,ⅡB2 andⅡB3 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. AllelesⅡB1*01,ⅡB2*01 andⅡB3*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 MHCⅡB. 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.更多还原