Research On The Discipline Of Geological Genetic Diversity Of Tripterygium Wilfordii Hook.F' Provenances

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...