| Tetrastigma hemsleyanum Diels et Gilg,commonly known as Sanyeqing,is a perennial plant of the genus Tetrastigma in the family Vitaceae native to China.It has various medicinal properties,such as anti-inflammatory,anti-cancer,and anti-tumor effects,and is known as the “natural plant anti-biotic”.In recent years,the market demand for T.hemsleyanum as an important traditional Chinese medicine has increased.However,there are concerns about the inconsistent quality of medicinal herbs and the decreasing genetic diversity of the plant resources.Additionally,there needs to be a comprehensive and systematic evaluation system for the genetic resources in the production and acquisition of T.hemsleyanum.In this study,the samples were collected from five different provinces in China,mainly from Zhejiang and from the main production areas of four other provinces,and their phenotype,medicinal components,stress resistance,and genetic information were comprehensively evaluated.We screened out superior strains with excellent characteristics and studied the genetic diversity level of the germplasm resources.We established a technology system for the comprehensive evaluation of the germplasm resources based on RAD-seq,morphological,physiological,and biochemical characteristics,as well as medicinal components.This system will help to protect and utilize the genetic resources of T.hemsleyanum providing a scientific basis for the breeding and quality control of this important traditional Chinese medicine.The main research results are as follows:(1)Research evaluating of T.hemsleyanum germplasm resources based on phenotype traits.The agronomic traits of the aboveground and underground parts of 23 different strains of T.hemsleyanum from different regions were measured,and the morphological differences among T.hemsleyanum from different regions were compared.The results showed that the leaf and tuber morphology of T.hemsleyanum from different regions varied.Leaf color was divided into green and purple,and leaf shapes were mainly ovate,lanceolate,ovoid,acuminate,and ovate-oblong.Leaf margins had fine and coarse serrations.Tuber shapes were bead-like and ovoid.Meanwhile,there are significant differences in various agronomic traits among populations.Furthermore,further studies have revealed significant differences among different strains of Zhejiang.In order to further understand the genetic stability and improvement potential of T.hemsleyanum,a variation analysis was conducted on each trait.The results showed that the coefficient of variation of each agronomic trait was above 20%,indicating that each trait had significant genetic potential and abundant genetic diversity,providing abundant parent materials for the screening of superior strains in the future.(2)Evaluation study of T.hemsleyanum germplasm resources based on active ingredients.By determining the active ingredients and index components of the aboveground parts of 23 different strains of T.hemsleyanum from different regions and the active ingredients and index components of the underground parts of 16 different strains of T.hemsleyanum from different regions,the chemical basis of T.hemsleyanum was preliminarily explored in order to evaluate the medicinal value of the entire plant and screen for superior strains.The principal component analysis results showed that the FJXL strain,the ZJWZ strain,and the HNJS strain ranked the top three in the comprehensive scores of active ingredients in the aboveground parts of plants among different provinces,the top-ranking strains are ZJWZ,ZJLS02,and ZJKH in T.hemsleyanum from the Zhejiang province;while the ZJCA strain,ZJTZ strain,and ZJLA01 strain from Zhejiang Province ranked higher in the comprehensive scores of the underground components.Therefore,the ZJCA strain,ZJTZ strain,ZJLA01 strain,ZJLS02 strain and ZJKH strain were selected as candidate superior breeding strains in T.hemsleyanum from the Zhejiang province.Meanwhile,there were significant differences in index components of T.hemsleyanum from different regions,and the strains with higher total content of index components were HNJS,HNCS strain from Hunan Province,and ZJCA strain from Zhejiang Province.(3)Research on the evaluation of T.hemsleyanum germplasm resources based on genetic information.A total of 67 germplasm materials from different regions were analyzed to obtain population SNP sites by using RAD-seq technology.The study aimed to explore the genetic diversity of T.hemsleyanum germplasm resources by estimating the level of population genetic diversity,analyzing population genetic structure,studying phylogenetic relationships,and conducting principal component analysis.The results showed that the results of population structure analysis,principal component analysis,and phylogenetic analysis were highly consistent and mutually corroborating.The population genetic structure of the 67 germplasm materials was divided into four subgroups,with some hybridization occurring within each subgroup.Furthermore,there was a certain degree of gene flow between subgroups.This may be due to various factors such as seed dispersal,breeding system of the species,geographical distribution,geological changes,and historical climate.The genetic differentiation of T.hemsleyanum indicated that the degree of genetic differentiation between the four subgroups was high,with significant genetic differences and rich genetic diversity.These findings are conducive to the exploration of superior parental lines and provides an important material basis for breeding and improving of T.hemsleyanum in the future.(4)Research on the evaluation of T.hemsleyanum germplasm resources based on stress resistance.Based on the preliminary research results,different T.hemsleyanum strains from five provinces were selected and subjected to various abiotic stress treatments(cadmium,salt,and low temperature).Physiological and biochemical indicators,effective component contents,and related gene expression levels of T.hemsleyanum were measured to assess their growth status and adaptability under stressful conditions.The results of quantitative real-time PCR showed that the trend of changes in gene expression levels of the target gene was consistent with the trends in APX activity,SOD activity,total flavonoid content,and polysaccharide content for each strain.In addition,the genes related to the biosynthesis of flavonoids were positively correlated with total flavonoid and total phenolic content to varying degrees.The genes related to the biosynthesis of polysaccharides were weakly positively correlated with polysaccharide content.Through subordinate function analysis,it was found that the ZJWZ strain from Zhejiang Province had the highest D value,the best overall performance,and the most vigorous stress resistance.Under adverse conditions,its antioxidant enzyme activity,total chlorophyll,carotenoid,and effective component content increased,demonstrating better growth status and adaptability.It is indicated that the ZJWZ strain from Zhejiang Province can be used as the preferred parental material for subsequent resistance breeding. |
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