【作者】 范文洁；

【导师】 吴承祯； 洪伟；

【作者基本信息】 福建农林大学 ， 自然地理学， 2010， 硕士

【摘要】 植物地理学研究的重点是植物的起源、演化与空间分布的关系,主要依托于植物在某一地理区域内表现出的规律性。这种规律可以是外在形态,也可以是内在特异基因。本研究以药用植物雷公藤(Tripterygium wilfordii Hook.F)为具体研究对象,通过对不同种源及个体的遗传多样性和遗传结构的研究,探讨地理差异和人为干扰对雷公藤遗传分化的影响,最终对雷公藤种质资源保存提出建议。从雷公藤的主要分布区福建、江西、浙江、湖南、贵州、云南、广西7省采集95个个体用于RAPD分子标记实验,并结合居群采样原则和空间自相关分析提出雷公藤迁地保护策略。研究成果如下:1、将提取植物总DNA的常规CTAB法和试剂盒法结合,所得方法能够成功地从雷公藤叶片中分离出纯度高、杂质少的优质DNA。该方法排除了以往提取雷公藤DNA时多糖和酚类物质的干扰,且适用性较强。同时通过多种提取方法的试验结果对比,发现雷公藤内含物中存在某种物质能够中和提取液,从而干扰提取结果,但该物质能通过氯仿抽提消除或是减少。2、筛选出12条随机引物对全部供试样本进行RAPD分析,一共得到118个重复性好、清晰的位点,其中多态位点共计107个,多态百分比达到91.14%;全体样本Nei’s基因多样性指数H=0.2973,Shannon’s信息指数I=0.4472,观测等位基因数Na =1.9114,有效等位基因数Ne =1.5120。4项衡量居群遗传多样性的重要指标均显示供试25个群体具有较高的遗传多样性水平;东南部种源Nei’s基因多样性指数和Shannon’s信息指数普遍高于西南部,主要是因为东南部为雷公藤制药优质药材产区,开发利用较早,杂交和引种加强了种源间的交流,而西南部种源多被高山包围,地理上不利物种间基因交流。3、利用两种软件对供试材料进行亲缘聚类。Popgene聚类结果为广西、贵州、云南3省种源聚集在一起形成固定的西南居群区;福建种源分别和浙江、江西、湖南三省种源聚集成3个居群区。总体上看,东南地区基因交流密切,且江西、湖南两省为雷公藤和昆明山海棠的混生区,因而种源间亲缘关系较复杂。DPS聚类结果基本一致,总体保持了先个体聚集后地区聚集的态势。但福建居群明显独立,混生区聚类也更趋合理。4、制定雷公藤种质资源迁地保护策略,需要同时考虑居群内取样数、居群数和遗传变异空间关系。基于对雷公藤遗传多样性的各项研究得出,以保存遗传多样性为主要目的的雷公藤迁地保护,每个居群的采样数需达到45才能充分代表该居群的遗传多样性;同时,不同地区的取样居群数不同,西南地区取到4个居群就能保证包含群体95%的遗传多样性,而东南地区最多需取到10个居群。对居群遗传变异空间自相关分析的结果为,在较近距离等级(738.4m)内,个体间存在相似关系,但随着距离的增大,Moran’s I值中正值减少而负值增多,特别在第六等级距离内出现了明显拐点,说明在1044-2350m范围内的个体不相似。尽管在后两个等级中又出现了少数正相关的Moran’s I值,但基于正负值总体走势,依然反应出负值的渐变模式。5、将生长于永安国有林场的18个种源的360个个体在生长8个月后的生物量增值和器官生长各项指标作因子分析,最终得出浙江丽水、广西柳州、三明明溪、浙江衢州、浙江金华、三明清流、福建莆田、三明尤溪等地种源在永安的生长情况最好,且除去广西柳州和福建莆田,其余6个种源均被归为同一遗传分支。这说明遗传距离相近的种源,对生长环境的需求也相近。而作为本地种源,永安种源的得分处于平均水平之下,表明永安供试种源在本地并不具备生存优越性。因此,在制定保护策略时可将亲缘关系相近的种源移植到一起,同时要确保迁地保护区为雷公藤的最佳生境。更多还原

【Abstract】 The key point of Phytogeography research is the relationship of the origin, evolvement and spatial distribution of plants which is based on the rule of plants performed in a particular geographical area. This rule could exist in the external forms, and also could be in some internal special gene. This paper took Tripterygium wilfordii Hook.F as a specific research subject, and the affection of the genetic diversity of Tripterygium wilfordii Hook. F caused by geographic distribution and human disturbance was studied based on the research of the genetic diversity and genetic structure of the individual Tripterygium wilfordii Hook.F with different provenances, and finally the conservation principle and methods of the germplasm resources of Tripterygium wilfordii Hook.F were proposed. 95 samples have been collected from seven provinces, the main distribution areas which were Fujian, Jiangxi, Zhejiang, Hu’nan, Guizhou, Yunnan and Guangxi, and the samples were used to RAPD molecular marker experiments. The genetic diversity and genetic structure of Tripterygium wilfordii Hook. F population has been concluded, and the strategy of off-site conservation of Tripterygium wilfordii Hook. F was presented combined with the collection principles of wild Tripterygium wilfordii Hook. F samples and the analysis of spatial autocorrelation. The main results were as following:1. CTAB method and kits which were the typical vegetation general DNA extracting methods were combined together and being used successfully to separate excellent quality DNA of high purity and fewer impurities from the leaves of Tripterygium wilfordii Hook. F. This technology excluded the disturbance of high levels of polysaccharides and polyphenol which was a particularly difficult problem due to the high level of those before, and also the technology had strong applicability. At the same time from the experimental results comparison of different extracting technologies, found that something in the body of Tripterygium wilfordii Hook. F could neutralize the extracting solution and then influenced the extraction result, but the material could be extracted by chloroform to reach a lower level.2. RAPD of all tested samples were analyzed by 12 random primers chosen, and 118 gene locus were acquired which had good repetition of experiment and clear locus. The ratio of polymorphic loci was 91. 14% including 107 loci, and the Nei’s gene diversity index H was 0.2973, information index of Shannon’s was I=0.4427, the observed number of alleles was 1.9114, and effective number of alleles was 1.5120. The four important gene diversity indexes all showed that the 25 tested groups had relatively high gene diversity. The Nei’s gene diversity index and Shannon’s information index of Tripterygium wilfordii Hook. F provenance in southeast area was generally higher than those in southwest area. The reason of this result was that the high quality raw materials of Tripterygium wilfordii Hook. F for Chinese medicine were mostly from southeast of China and the development and utilization of Tripterygium wilfordii Hook. F was relatively earlier, and the hybridization and introduction of this plant has improved the communication of provenances, however the provenances in the southwest area were mostly surrounded by high mountains which was disadvantage in geographical location for the species’gene communication.3. Clustering analysis of phylogenetic relationships of testing materials was made using two soft wares. The clustering result of Popgene was that provenances of Guangxi, Guizhou and Yunnan three provinces gathered together which formed the regular southwest population area; provenances of Fujian formed three population living areas with provenances of Zhejiang, Jiangxi and Hunan provinces. In general, gene communication in southeast area was very closely, and Jiangxi and Hunan provinces were mixed living areas of Tripterygium wilfordii Hook. F and Tripterygium hypoglaucum(levl)Hutch, so the relation of provenances was very complicated. The results of DPS cluster were very similar and firstly individual aggregation and latter area aggregation generally. But the population in Fujian province was obviously independent, and the cluster in mixed living area was becoming more reasonable.4. Making the strategy of off-site conservation of Tripterygium wilfordii Hook. F gene resource, you need to consider the sample size, population number and spatial relations of genetic variation. the research basing on the genetic diversity of Tripterygium wilfordii Hook. F showed that the off-site conservation whose principal object was protecting genetic diversity required that in order to well represent the genetic diversity of the population the sample size need to reach 45, and meanwhile the population sample size was different according to different areas. In the southwest area, 4 population samples was enough to ensure including 95% genetic diversity level, while 10 population samples were required in southeast area at most. The result of the spatial autocorrelation analysis of genetic variation was that there were similar relationships among individuals within the nearest distance scales (783. 4m), but the number of positive value of Moran’s I value decreased and negative value increased especially exist an obvious inflexion in the sixth distance scale with the increase of the distance which indicated that the individual was not similar within the range of 1044-2350m. Although minority positive correlation of Moran’s I value existed in the latter two scales, in general of the overall trend of all values, it still indicated the pattern of incremental rule of negative value.5. The factor analysis was conducted based on the biomass increase amount and organ growth indexes of 360 individuals including 18provenances which were planted in Yong’an state-owned forest center, and finally reached a conclusion that the Tripterygium wilfordii Hook. F population from different provenances sites of Zhejiang Lishui, Guangxi Liuzhou, Sanming Mingxi, Zhejiang Quzhou, Zhejiang Jinhua, Sanming Qingliu, Fujian Putian and Sanming Youxi growed very well. Other 6 provenances were classified into one genetic branch except that in Guangzhou Liuzhou city and Fujian Putian city which indicated that the environmental requirement of the population provenances with the similar genetic distance was also very similar. and as the local provenance population, the score of Tripterygium wilfordii Hook. F in Yong’an city was below average level which showed that the provenance tested in Yong’an city had no survival superiority than others. When making the strategy of protection, we can transplant Tripterygium wilfordii Hook. F populations of similar provenances and at the same time make sure that the off-site conservation area has the best growing environment for Tripterygium wilfordii Hook. F更多还原