基于DNA分子标记的黄缘闭壳龟遗传多样性与种群遗传结构研究

发布时间：2018-11-10 20:04

【摘要】：河南信阳大别山区是黄缘闭壳龟最重要的分布区之一,但是由于生境破坏、环境污染和滥杀滥捕等原因,黄缘闭壳龟的野生资源量急剧下降,目前已被IUCN及CITES列为珍稀濒危物种,濒危等级EN。为了给黄缘闭壳龟提供在分子水平上的理论依据,本研究对黄缘闭壳龟进行了微卫星标记、单核苷酸多态性标记的开发,对黄缘闭壳龟卵壳膜与粪便进行了非损伤DNA试提取,并利用微卫星标记和线粒体标记技术对黄缘闭壳龟的群体进行研究。通过对黄缘闭壳龟群体的遗传多样性和遗传结构进行分析,测定群体间的遗传差异,预测杂种优势,为种质资源库的构建提供理论基础,同时也为大别山黄缘闭壳龟野生资源的保护和人工养殖的开展提供建议。本文研究的主要结果有:1.本研究采用FIASCO法,构建了黄缘闭壳龟富集文库,成功分离设计得到136对微卫星引物,并对微卫星位点进行评价,共检测出44对多态性微卫星引物。2.尝试采用磁珠富集的方法对濒危保护动物黄缘闭壳龟基因组中潜在的SNP位点进行了开发,共获得10条确认SNP位点的引物序列。3.以幼龟孵化后残留的卵壳和新鲜的粪便为研究材料,采用非损伤性方法对黄缘闭壳龟基因组DNA进行提取,证实了从粪便中提取DNA是不可行的,而从卵壳膜中提取DNA是可行的,能为后续黄缘闭壳龟的分子遗传提供研究材料。4.利用新开发的微卫星标记对3个不同来源的43只黄缘闭壳龟群体的遗传结构进行分析,发现养殖场中部分个体出现了不同亚群之间的基因交流现象,并将救护中心类群和师院类群合并为一个亚群,养殖场的两个类群分别作为一个亚群。5.利用线粒体基因CO1和CO2对来3个不同来源的黄缘闭壳龟群体(21只)进行了单倍型和系统发育分析,结合微卫星标记分析结果,把所有个体分为信阳地区种群,安徽地区种群和未知地区种群三个不同的种群,并推测未知地区种群可能为大别山以外地区的种群且与其他个体存在较远的进化关系,而信阳地区种群和安徽地区种群之间的进化关系更近。
[Abstract]:The Dabie Mountain area of Xinyang, Henan Province is one of the most important distribution areas of the closed shell tortoise. However, due to habitat destruction, environmental pollution and indiscriminate hunting, the wild resources of the closed shell tortoise have declined sharply. At present, it has been listed as rare and endangered species by IUCN and CITES, and EN. has been classified as endangered. In order to provide theoretical basis on molecular level, microsatellite markers and single nucleotide polymorphism (SNP) markers were used to extract DNA from egg shell membrane and feces. Microsatellite markers and mitochondrial markers were used to study the population of the closed shell tortoise. Based on the analysis of genetic diversity and genetic structure of the populations, the genetic differences among populations were determined, and heterosis was predicted, which provided a theoretical basis for the construction of germplasm resource bank. At the same time, it also provides suggestions for the protection of wild resources and the development of artificial breeding of the closed shell turtle in the yellow margin of Dabie Mountain. The main results of this paper are as follows: 1. In this study, FIASCO method was used to construct the enrichment library of the turnip, and 136 pairs of microsatellite primers were designed successfully, and the microsatellite loci were evaluated. A total of 44 pairs of polymorphic microsatellite primers were detected. In this paper, the potential SNP loci in the genome of endangered protected animals were developed by means of magnetic bead enrichment. A total of 10 primer sequences were obtained to confirm the SNP loci. Using egg shell and fresh feces from hatching of young turtle as the research materials, the genomic DNA was extracted by non-invasive method. It was proved that extracting DNA from feces was not feasible, but DNA from egg shell membrane was feasible. It can be used to study the molecular heredity of the subsequent turnip turtle. 4. Using the newly developed microsatellite markers, the genetic structure of 43 populations from three different sources was analyzed. It was found that some individuals in the farm had the phenomenon of gene exchange among different subpopulations. The rescue center group and the teacher's college group were merged into one subgroup, and the two groups in the farm were regarded as one subgroup respectively. Haplotype and phylogenetic analysis were carried out in 21 populations (21 individuals) from three different sources of yellow-bordered turtle by mitochondrial gene CO1 and CO2. Combined with the results of microsatellite marker analysis, all individuals were divided into two populations in Xinyang area. There are three different populations in Anhui and unknown regions, and it is speculated that the population in the unknown region may be the population outside the Dabie Mountains and has a far evolutionary relationship with other individuals. The evolutionary relationship between Xinyang population and Anhui population is closer.
【学位授予单位】：信阳师范学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q953

【参考文献】