武陵山区蛇足石杉遗传多样性与石杉碱甲含量相关性研究

In this project, we are going to investigate the genetic diversity and genetic structure at the population level for Huperzia serrata occurred in Wuling Mountain range using AFLP and SSR markers. Combining with the garden introduction experiment, HupA content of the living plants,which are in one to one corresponding to those samples used in genetic diversity experiments, are determined dynamically by HPLC method.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 assess the gene flow mediated by spores. 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 relation between genetic diversity and HupA content is also to be analyzed and illustrated. Based on the genetic variation and structure revealed by the AFLP and SSR markers, combining with the results of the study for the breeding system, the current situation of the population genetic structures and their evolving trend in future will have been analyzed. Finally, the relationship between the genetic variation and some factors such as habitat disturbance and clonal growth will have been clarified. Thus, the effective conservation and sustainable utilization strategy for Huperzia serrata germplasm resources could be put forward.

以AFLP、SSR分子标记和HPLC方法为主要手段，结合同园引种试验，对武陵山区不同样地的蛇足石杉居群进行遗传多样性研究和石杉碱甲含量动态跟踪测定。将基于SSR分子标记进行的亲本分析和野外观察有机结合，探明各居群的自（异）交率，确定居群内的交配格局；探讨遗传分化与基因流之间的关系，以及遗传变异在居群水平上的分配；分析阐明石杉碱甲含量与遗传多样性之间的相关性；基于AFLP和SSR所揭示的遗传变异和遗传结构，结合繁育系统的研究结果对居群遗传结构现状和未来变化趋势进行分析预测，并探讨生境扰动、克隆生长等因素与遗传变异和遗传结构之间的关系，为科学制定并实施蛇足石杉种质资源的有效保护措施和持续利用策略提供基础理论依据。

本项目以分布于武陵山区15个样地的蛇足石杉居群为研究对象，运用AFLP和SSR分子标记技术及建立不同器官转录组并测序，对各蛇足石杉居群繁育系统、遗传多样性水平、结构与变异进行研究；通过同园引种栽培，对各居群植株石杉碱甲、乙、丙含量动态采用HPLC跟踪测定。基于AFLP和SSR实验所揭示的遗传多样性特征和对蛇足石杉根际土壤微生物多样性及土壤矿质元素含量的分析，探究遗传基因和环境因子多样性对蛇足石杉生理生态和次生代谢物质积累的影响以及它们之间的相关性。结果如下：.1.蛇足石杉遗传多样性在不同居群间有明显差异；居群间的遗传分化较低，而居群内的遗传分化极为显著；不同居群间的遗传距离与地理距离不呈正相关。遗传多样性与结构主要决定于居群历史，较少干扰且稳定的居群偏向克隆生长，遗传多样性较低；而新建居群则较高。克隆生长、生态位选择、异交及有效的孢子风媒传播等因素维持着高的遗传多样性水平，过度采挖等人类活动和生境片断化是导致蛇足石杉濒危的主要原因。.2.以典型样地植株不同器官建立转录组并进行测序，总共获得77,430,786 对高质量的reads，包括32,418,231,517对碱基；序列从头组装获得621,023 个contigs。基于公共数据库对49,923个unigenes进行了注释。根据KEGG数据库，集中挖掘出960个与植物代谢相关的unigenes，经代谢网络路径及分子功能分析，确定这些unigenes参与了蛇足石杉的初级代谢、能量代谢、碳代谢、植物生长以及与次生代谢物的生物合成等相关。该工作为了解石杉碱甲代谢途径及其生物合成机制，积累了重要参考数据。.3.对各样地植株在同园引种前后测定其石杉碱甲、乙、丙含量并进行比较分析，发现近90%的植株在引种移栽之后，2~3种石杉碱含量均明显下降，仅个别植株3种石杉碱含量同时升高。由此推测：生境条件或许比遗传因素更大程度影响石杉碱的生物合成与积累。建议将在遗传基因和对生境适应性都具有特殊优势的个别植株确定为优先保护对象，对其开展引种驯化及遗传和生理生态特性的深入研究，以期培育出优质高产的蛇足石杉新品系。