景观格局与生态过程对川东獐牙菜的基因流及微进化的影响

Swertia davidii Franch is an endemic plant of China which is distributed in the riparian zone of Wuling Mountain range and Three Gorges Valley of the Yangtze River. In this project, we are going to investigate the genetic diversity and genetic structure within and among the populations of S. davidii by establishing highly polymorphic DNA microsatellite markers (SSR). The parentage analysis based on the SSR markers is well integrated with field observation on the breeding system, so as to get the selfing rate, confirm the mating patterns within population, and the combination of selfing frequencies and paternity analysis will help us to test the pollen-mediated and seed-mediated gene flow. Moreover, the relationship between genetic differentiation and gene flow, as well as the distribution of the genetic variation in the population level will have been discussed.The adaptability and their evolving trend in future of the population to environment also will have been analyzed. Depending on landscape ecology principles, we will explore gene flow in one single river and among several rivers. The effects of environmental gradients on gene flow will be revealed by landscape patch and matrix. The results can provide a research case for landscape genetics and enrich some theory. At the other hand, the results will be a theoretical basis for drawing up the effective conservation and sustainable utilization strategy of this species.

川东獐牙菜为龙胆科獐牙菜属植物，具有重要的药用价值，分布在武陵山区河谷两岸，现存种群数量极少。本项目拟通过开发川东獐牙菜SSR分子标记，分析其种群内和种群间的遗传多样性和遗传结构，并将SSR分子标记进行的亲本分析与野外观察结合，进行植物种群的自交率测量及亲本分析，定量测定花粉和种子介导的基因流，分析种群对环境变化的适应能力和进化潜能。研究尺度从单一河谷内研究基因流扩散格局到不同河谷间比较基因流扩散格局，并引入景观生态学理论，考虑现实景观的复杂性与不确定性，用环境梯度取代关于斑块和基质的均质概念，更真实地反映景观结构与生态过程( 如河流、生境片断化、人为采挖、水淹等)对种群基因流的影响，研究结果不仅可丰富分子生态学的研究内容，为景观遗传学理论发展提供研究实例，同时为药用植物川东獐牙菜残存种群的遗传保育和种群复壮策略提供理论依据，并充分认识这一物种的生态遗传适应机制。

川东獐牙菜为龙胆科獐牙菜属植物，具有重要的药用价值，分布在武陵山区河谷两岸，现存种群数量极少。繁育系统研究对保护遗传多样性具有重要意义，是决定植物种群遗传结构的最主要因素。本研究表明该植物繁殖期一般从10月中旬至次年1月中旬，单花期约17d，花具有雌雄异熟和异位花结合的特征，繁育系统属于异交，部分自交亲和，需传粉者。有性生殖过程正常，花药4室，花药壁为基本型，成熟花粉为2-细胞，雌配子体发育为蓼型，胚乳发育为核型，胚胎发育为茄型，果实成熟时，种子发育至心形胚阶段。构建了川东獐牙菜的转录组数据库，进行生物信息学分析，在40对SSR引物中，32对引物均能扩增出可重复，清晰的条带，其中12对引物表现有多态性差异。每对引物扩增的位点数 (Na)平均为9.64，有效位点数(Ne)平均为3.56，Shannon指数（I）平均为1.62。SSR数据分析结果表明，川东獐牙菜遗传分化系数很高，Fst为0.5737，基因流Nm为0.1857，表明遗传变异主要存在于种群间，种群存在极大的遗传分化。聚类分析表明，沿乌江河域的种群先聚为一类，茅岩河域的种群聚为一类，剩余两条河流的种群聚为一类，然后各聚类群再聚集。红外光谱与紫外光谱原始图谱难以区分不同种群，FTIR与PLS-DA和HCA方法结合对川东獐牙菜进行种群（产地）鉴别的方法切实可行，紫外光谱结合主成分分析和偏最小二乘判别分析能够较好的鉴别不同种群（产地）川东獐牙菜，构建模型预测效果较好。光照有利于川东獐牙菜种子的萌发，其萌发最佳温度为20℃，GA3处理最佳方式为100mg/L浸泡12h，可显著提高种子发芽率和发芽势。以川东獐牙菜无菌种子苗为外植体，筛选最佳继代培养基为MS+ BA2.0mg/L+ NAA0.05mg/L，生根培养基最佳配方为1 /2 MS +BA1.0mg/L+ NAA 0.1mg /L+ kt 0.3mg/L。研究结果为药用植物川东獐牙菜残存种群的遗传保育和种群复壮提供理论依据。