Study On Germplasm Resources And Genetic Diversity Of Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge is one of the most important traditional Chinese medicinal plants belonging to the Lamiaceae family and has been used in China and some Asian countries for thousands of years. There are two major groups of biologically active components in S. miltiorrhiza, tanshinone compounds and phenolic acids. Its roots are widely used in several therapy systems for the treatment of angina pectoris, atherosclerosis, chronic renal failure, liver fibrosis, coronary heart disease and myocardial infarction. As a traditional Chinese medicine, Salvia miltiorrhiza Bunge has a long cultivated and applied history. However, there are scare of knowledge of genetic diversity among different germplasm, and there are few reports about the construction of germplasm resources library and genetic diversity of different S. miltiorrhiza accessions, which came from different morphological accessions and spaceflight induced accessions. In this study, S. miltiorrhiza individuals from the second generation of space induced, the third generation of space induced, different morphological characters and nine geographically wild populations in China were analyzed. Active constituents and root yield of the second generation of S. miltiorrhiza induced by spaceflight were preliminary studied. We constructed a germplasm resources library of S. miltiorrhiza that contained 62 SP3 accessions, 53 different morphology accessions, 6 wilds, 6 cultivars and 6 ground controls, the genetic diversity of 133 different accessions were studied based on morphological characters, isozymes, molecular markers, metabolic profiles and antioxidant activities. Moreover, we used two single primer amplification reactions to analyze the genetic relation between nine wild S. miltiorrhiza populations. This paper would benefit for the construction of germplasm library, collection of the germplasm resources, and also could provide a theoretical basis for the utilization and development of S. miltiorrhiza. The main results were as follows:1. Here, we surveyed the root yield and active constituents of the second generation of S. miltiorrhiza(SP2) responded to the spaceflight environment. High-performance liquid chromatography(HPLC) was conducted for the comprehensive constituent characterization of 28 SP2 lines(224 individuals) and the ground controls(eight individuals). The resultsshowed that the fresh and dry weight of roots displayed a span of 459.09 g and 94.17 g in SP2 lines. What’s more, the mean contents of four lipophilic compounds(tanshinones I, tanshinones IIA, cryptotanshinone and dihydrotanshinone) of 28 SP2 lines varied from 0.32 to 1.04 mg g-1, 0.47 to 2.39 mg g-1, 0.25 to 1.60 mg g-1, and 0.53 to 1.67 mg g-1. Except salvianolic acid B varied drastically from 71.96% to 201.37% of the ground control treatment, other six hydrophilic active constituents content of 28 SP2 lines all increased after spaceflight. Principal component analysis(PCA) was performed to obtain an overview of the distribution of all samples, and scores plot clearly separated the SP2 accessions from the ground controls.2. We constructed a germplasm resources library of S. miltiorrhiza that contained 62 SP3 accessions, 53 different morphology accessions, 6 wilds, 6 cultivars and 6 ground controls. We used 11 quality characters and 20 quantity characters to analyze the variations in morphological characters among 133 S. miltiorrhiza accessions. The results showed that there were extensive variations among them, the diversity index ranged from 2.87 to 4.88. Among the quality characters, the variance mainly lay in leaf color, leaf margin type, leaf fuzz, stem color and flower color; among the quantity characters, the variance mainly lay in plant height, ramification number, flower ring number per inflorescence, flower width and flower length. Based on UPGMA analysis of euclidean distance among individual accessions of S. miltiorrhiza, cultivated accessions and wild accessions shared a close relationship, the others were scattered.3. Three antioxidant enzymes of 133 S. miltiorrhiza accessions were analyzed. Based on the experiment result, we found that peroxidase(POD), superxide dismutase(SOD), and catalase(CAT) activities of the different morphology S. miltiorrhiza showed variation. The coefficient of variation(CV) ranged from 12.4%(SOD) to 45.3%(CAT) and all of the diversity index of these enzymes were above 4.5. We used polyacrylamide gel electrophoresis to analyze five isozymes(POD, CAT, esterase-EST, malic dehydrogenase-MDH, and alcohol degydrase-ADH) from leaves of all samples. And 30 isozymes loci, 25 polymorphic loci were observed, the percentage of polymorphic loci was 83.33%. According to the UPGMA cluster analysis, all samples were divided into seven clusters. The results indicated that antioxidant enzymes activities and five isozymes from leaves had apparent differencein S. miltiorrhiza germplasms.4. The genetic diversity of S. miltiorrhiza germplasms was studied by ISSR and SRAP markers. The results indicated that the average numbers of polymorphic bands amplified by per ISSR and SRAP primer were 13.6 and 41.6, respectively. The percentage of polymorphic loci at inter-species level was 94.5% and 92.3%, which revealed abundant genetic diversity on species level. Genetic diversity analysis based on integrated data of ISSR, SRAP andISSR+SRAP showed that the pedigrees revealed by them were basically similar i.e., all accessions were divided into two clusters. Cluster I comprised 115 accessions(S1-S115) and the different morphology accessions(S1-S53) and the SP3 accessions were clustered into one category on a specific genetic similarity coefficient, respectively. Cluster II included all cultivated, ground control and wild accessions. Among them, cultivates and ground controls were higher in genetic identity which was nearer in genetic relationships, the genetic distance between wilds and the two above populations was also near therefore the three populations could be clustered into one group. All accessions could be distinguished from each other according to a specific genetic similarity coefficient.5. High performance liquid chromatography was performed to determine metabolic profiles of shoots and roots of different S. miltiorrhiza germplasms. Results showed that the seven phenolic acids contents and four tanshiones contents were high in shoots and roots of different accessions and were tested with great variation among them, the CV of active constituents ranged from 15.8% to 47.9% in shoots and 24.7% to 67.1% in roots. Our study indicated that the content of active constituents may be affected by morphological characters and spaceflight.6. UV-detected method was performed to determine the contents of total phenolics and total flavonoids. Meanwhile, the antioxidant activity of extract of shoots and roots of all samples was evaluated by four different antioxidant assays(DPPH, ABTS, FRAP and CUPRAC). Results showed that shoots and roots were abundant in total phenolics and total flavonoids; variation existed among different accessions, the CV varied from 24.1% to 40.0%. The extract of shoots and roots revealed high antioxidant activity of DPPH, ABTS, FRAP and CUPRAC. Our study implied that S. miltiorrhiza has powerful antioxidant activities and different accessions present considerable variation.7. Genetic variability in the wild genotypes of Salvia miltiorrhiza from different parts of China was analyzed using two remarkable single primer amplification reactions(SPAR) markers, viz., Inter-simple Sequence Repeats(ISSR) and Start Codon Targeted(SCo T). One hundred and eighty eight(182) and Two hundred and seventy six(276) amplification fragments were scored by 13 ISSR primers and 20 SCo T primers, respectively. The percentages of polymorphic loci of ISSR marker and SCo T marker were 90.11% and 85.14%, which indicated that there were abundant polymorphisms in Salvia miltiorrhiza germplasm. Analysis of molecular variance(AMOVA) of pairwise distances indicated both high variation at intra-population level(46.26%) and inter-population level(53.74%). Comparing with percentage of polymorphic loci of nine populations, genetic diversity of Yuncheng population was the highest, while Rizhao population was the lowest. Similar clusters were obtained bycluster analysis using the unweight pair-group method with arithmetic average(UPGMA), indicating that Yuncheng population and Shangluo population showed the closed relationship, Ruijin population was a relatively independent from the other populations. Mantel test demonstrated that there no correlationship between genetic distance and geographic distance matrices.8. In conclusion, our results demonstrated that the germplasm resources library of Salvia miltiorrhiza showed rich genetic diversity by systematic study and evaluation. The different S. miltiorrhiza accessions can effectively distinguished by the selected evaluation markers. The morphological traits, physiological and biochemical level, molecular markers, active constituents and antioxidant activities of cultivated, wild and ground control S. miltiorrhiza gerplasm, which are the three more stable populations were expressed a certain degree of stability. However, the different morphological S. miltiorrhiza accessions and the SP3 accessions existed large variation level among them. Comparison of cluster analysis based on molecular markers and the active constituents showed that there is a big similarity between molecular markers and active constituents of different S. miltiorrhiza accessions, suggesting that the active constituents in S. miltiorrhiza was relevant to its heredity. Based on the results of correlation analysis between morphological traits and other markers revealed that there are correlations between the morphological traits and physiological activity, active constituents and antioxidant activities of S. miltiorrhiza, which can provide a theoretical basis for directional selection of S. miltiorrhiza germplasm.