Study On Population Genetics In The Genus Gynostemma BL. (Cucurbitaceae)

Gynostemma BL., which belongs to the family Cucurbitaceae, contains 17 species and 2 varieties, which fall into 2 subgeneras, among them,9 species and 2 varieties are unique to China. Plants of the genus Gynostemma BL. grows in the edge of the forests, sparse woods or thickets. The polyploidy phenomenon exists in Gynostemma BL, especially in the most widely distributed species Gynostemma pentaphyllun (Thunb.)Makino. It has diploid, tetraploid, hexaploid and octaploid in cell types, but it was not obviously different in the morphology among individuals of different ploidy.Our study used 6 pairs of SSR primers, which were screened from 30 pairs, analyzed in 98 natural populations of 1430 individuals by PCR, tested the products with 12% native polyacrylamide gel electrophoresis, calculated the genetic parameters, and further then carried out the genetic diversity and species differentiation analysis. We focused on the analysis in 72 natural populations of 1043 individuals of G. pentaphyllun, discussed the genetic diversity, genetic structure, spread mode and the reasons of polyploidy distribution pattern. In order to understand the genetic background of the plants of the genus Gynostemma, and to give rise to theoretical foundation for its resources protection and rational utilization. Through our research, we summarized some results as following:1. In this study,77 alleles were detected from 6 pairs of primers. The alleles number were from 10 to 15 in different loci. The PIC value range from 0.111 to 0.854, mean value was 0.515. The genetic diversity of G pentaphyllun in 72 populations was also analyzed by the 6 pairs of primers. It shown that the genetic diversity of G. pentaphyllun was low:the average value of the Nei’s genetic diversity index (He), observed heterozygosity (Ho), Shannon index (Ⅰ) and percentage of polymorphic loci (PPL) was 0.217,0.286,0.343 and 62.73%, respectively. AMOVA analysis revealed that genetic variation mainly existed among the populations (among populations was 51.57%,within populations was 48.43%). Furthermore, it possessed significant population genetic differentiation (FsT= 0.408) and gene flow was limited (Nm= 0.608).2. The results of genetic relationship and genetic structure shown that the differentiation of natural populations of G. pentaphyllun was due to the influence of geographical isolation.72 populations could be divided into 3 clusters by STRUCTURE analysis, and the obvious genetic exchanges existed among them in parts of areas in geographical, which had a high level of genetic diversity. The outcomes of the potential distribution prediction indicated that the G pentaphyllun populations in The Hengduan Mountains in the southwest was old relatively. Therefore, we speculated that the G. pentaphyllun in China originated in this region and then divided into 3 clusters spreading eastward and northward. Nanling Mountains and Qin-Ba Mountain Areas were the secondary contact regions for populations spreading, integrating genes from different clusters in these areas.3. The polyploidization of G. pentaphyllun occurred in the long and wide range of migration and proliferation. Most of the polyploidy populations were probably originated from the adjacent diploid populations. For the polyploidy populations which are not belonged to the same clusters with the diploid populations nearby, we speculated that their polyploidization was a spontaneous process. Moreover, the genetic diversity of polyploidy G pentaphyllun populations is higher than the diploid populations in the mass. They showed a better adaptability.4. The microsatellite analysis in 32 populations of 483 individuals among different species in Gynostemma BL. showed a higher genetic diversity level in Subgen. Trirostellum than in Subgen. Gynostemma. The genetic diversity of all the species were not high in the species level, in which the richest genetic diversity was in hexaploid G. cardiospermum, diploid G microspermum and G. laxiflorum. and the lowest was found in G laxum, G. pentaphyllum var. dasycarpum and G. burmanicum.5. Clustering analysis was consistent with the PC A analysis in the genetic relationship among all the species in Gynostemma BL.. They suggested that the populations of Subgen. Trirostellum were in the same group without G. cardiospermum, G. yixingense and G. aggregatum which were as a set with Subgen. Gynostemma. Although Subgen. Gynostemma occupied 2 groups, the further species clustering relations still needed more researchs.6. At last, according to our research, we put forward the protection strategies for the plants of Gynostemma BL. We suggest that:(1) do the reasonable ex-situ conservation based on the in-situ conservation; (2) analyze the genetic characteristics for species belong to Gynostemma BL, rich the gene pool, protect the genetic diversity; (3) in order to restore the species’ population size, stop artificial damage such as felling trees unlawfully, exploiting forests excessively and wastefully and so on. (4) it is necessary to prevent the pollution from cultivated species to the natural populations.