| Gynostemma pentaphyllum(Thunb.)Makin,which belong to the family of Cucurbitaceae,a herbaceous perennial climbing plant,is an important economic crops in some areas of our country.Gynostemma pentaphyllum is widely geographic distribution and strongly adaptability to the environment in our country.With the damaged ecological environment in recent years,its wild resources reserves sharply decline,the genetic diversity of Gynostemma pentaphyllum germplasm resources have been seriously threatened.In this study,as much as possible to collect wild Gynostemma pentaphyllum germplasm,building Gynostemma pentaphyllum resources nursery,use SSR and ISSR markers research genetic diversity of those germplasms,aims to Gynostemma pentaphyllum germplasm identification,breeding and conservation provide theoretical basises.The experimental results of this study are as follows:1.The author through visiting survey and literature search,germplasm resources nursery is established in Pingli Contry of Shaanxi province based on the collection,reproduction and preservation of wild Gynostemma pentaphyllum germplasm resources.76 wild germplasm materials and some cultivated varieties of Gynostemma pentaphyllum were collected from 13 provinces which contains the main distribution area of Gynostemma pentaphyllum in China.The new germplasm resources nursery provides the reliable basis of genetic materials for biology research.We can choose characters of superior varieties in order to develop varieties breeding work,for the breeding of excellent new varieties.At the same time,it also to provide a good platform for conservation of the genetic diversity,sustainable utilization of Gynostemma pentaphyllum resources.2.A total of 63 putative alleles were generated from 6 pairs of primers,The number of alleles was from 4to 15 in different locus,with a mean of 8.29 per locus.The polymorphic information content(PIC)in the range of 0.176-0.867,and mean value was 0.549.The mean of the Nei's genetic diversity index(He),observed heterozygosity(Ho)and Shannon index was 0.262?0.328 and 0.364,respectively.The average genetic distance and genetic similarity coefficients among 77 germplasms varieties was 0.392 and 0.679,indicating a wide genetic basis between the resources.Based on similarity coefficient among resources,77 resources can be divided into five categories by UPGMA clustering analysis of all tested germplasms.The results of clustering and principal components analysis(PCA)suggested that the genetic relationship between 77 germplasm resources was more complicated.PIC,Nei's genetic diversity index(He,Ho),Shannon index(I)and Genetic similarity coefficient(GS)indicated that SSR markers can effectively reveal the genetic variation between those Gynostemma pentaphyllum germplasm materials,and the genetic diversity was considerably abundant within wild resources.3.Comparison of the SSR(simple sequence repeats)and ISSR(inter-simple sequence repeats)markers,two different DNA markers techniques in Gynostemma pentaphyllum germplasm resources research for genetic diversity.Allelic loci 57 were detected from five pairs SSR primers,with a mean of 11.4 per locus,and percentage of polymorphic loci(PPL)was 92.98%.The mean PIC value of each SSR primer was 0.562.214 discernible DNA fragments were generated by 15 ISSR primers,and 206(96.26%)fragments were being polymorphic,and an average PIC value of 0.618.Analysis show that the two techniques were able to effectively reveal the genetic variation,a higher genetic diversity and good consistency among Gynostemma pentaphyllum germplasm resources tested,but ISSRs presented greater information content.The mean genetic similarity coefficients among all germplasm resources genotypes ascribed by SSR and ISSR matrices were 0.718 and 0.691,respectively.Clustering results of two molecular markers have some differences.Similar correlation coefficient correlation test showed both markers positive correlation The correlation of similarity coefficients showed positive correlation for both markers marker systems used(r=0.7287,P<0.01)It could be inferred that the two DNA marker techniques,both of which are suitable for the study of genetic diversity in Gynostemma pentaphyllum genetic diversity research.4.Used ISSR markers analyzed the genetic diversities and phylogenetic relationships of 77 Gynostemma pentaphyllun Germplasms from 13 provinces in different ecological environments.The results showed that,18 primers were selected from 100 primers which had clear,stable polymorphic bands.A total of 253 loci were obtained,including 244 loci,polymorphic loci(PPL)was 96.44%.The mean observed number of alleles(Na),effective number of alleles(Ne),Nei's genetic index(H)and Shannon information index were 1.9727?1.5417?0.3262 and 0.4948,respectively.The genetic distance(GD)among the tested germplasms genotypes varied from 0.043-0.656,mean value was 0.404.The enetic similarity coefficient(GS)ranged from0.519-0.977,mean value was 0.671 which shows a wide gene pooland a higher genetic diversity among the materials of Gynostemma pentaphyllum germplasms.Cluster annlysis with UPGMA method showed that the 77 testing germplasms were divided into 5 groups at the level GS 0.680,the genetic relationships was not entirely consistent with geographical distribution and ecological environment.Principal component analysis(PCA)and cluster analysis wsa more consistent,and the genetic relationships among the tested Gynostemma pentaphyllum germplasms could be more clearly and directly presented by the principal component analysis.5.Molecular identification of 77 Gynostemma pentaphyllum germplasm resources was conducted by ISSR markers.The results showed that the absence of 17 unique ISSR markers and the the presence of 12 unique markers obtained from 9 primers made it could be able to identify GZ-AS,GZ-JP,YN-JH,YN-ML,etc.23 Gynostemma pentaphyllum germplasm resources.In addition,the band patterns combination of primer UBC807,UBC841,UBC891 and UBC895 could be provide the basis for effective identification of 77 germplasm resources,which was successfully used to build the DNA fingerprinting for discriminating 77 Gynostemma pentaphyllum germplasms resources. |
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