Studies On Genetic Diversity Of Germplasm In Schisandra Sphenanthera Rehd. Et Wils.

Schisandra sphenanthera Rehd. et Wils. is a frequently-used Chinese herb plant, Due to renewed emphasis on its medicinal value in recent years, its market demand has been rising year by year, resulting in a sharp decline of its wild resources. Currently, it has been incorporated into our National Key Protected Wild Medicinal Species List, and it is the third class conservation of wild medicinal species, namely, the severely reduced resource and meanwhile the major common species of wild medicinal herbs. As there is very little information about S. sphenanthera, their genetic diversity and genetic structure are still not clear, besides, there is also a lack of comparison and analysis about phenotypic traits, pharmaceutical composition and genetic variation among different populations. Thus, it has been more urgent to make effort to collect, preserve and analyze its germplasm resource. We also should make clear its genetic structure and its distribution patterns, so that we can protect the germplasm resource and make full use of it.In this study, S. sphenanthera germplasms from Henan, Shanxi, Gansu, Sichuan, Chongqing, Zhejiang, Anhui, Shaanxi, and in 12 populations in China are analyzed by morphological, phytochemical and molecular marker technology. Phenotypic diversities of 238 collected germplasm are analyzed, and lignins contents and AFLP molecular of 85 samples are marked. From morphology, lignins contents and DNA, genetic diversity of S. sphenanthera germplasm in China are analyzed. The main results are as follows:1.Among and within the twenty phenotypic traits of S. sphenanthera populations, there exist a highly significant difference (P<0.01),Hanzhong,Shangluo, Baoji, Gansu, Chongqing and Henan populations varied greatly, with the rich genetic diversity. Genetic diversity is mainly within populations (71.04%).The traits of phenotypic fruit are rich in diversity, especially signal fruit weight (SFW), berry longitudinal diameter (BLD), berry horizontal diameter (BHD) and 100 berries fresh weight (BFW) and other traits in the great variation within populations, while the fruit shape index (FSI), longitudinal diameter of fruit (LDF), fruiting pedicels length (FPL) and berry numbers of signal fruit (BNSF) in large within population variation. Principal component analysis revealed that, BLD, BHD, BFW,and LDF, FSI, leaf length of new branch (LLNB), leaf width of new branch (LWNB), leaf length of old branch (LLOB), leaf width of old branch (LWOB) and so on, are the main factors of the phenotypic variation. UPGMA cluster devides 12 kinds of clustering into 4 types, Sichuan, Zhejiang and Anhui is one type, and the remaining nine kinds of cluster are a category.2.The phenotypic variation of S. sphenanthera fruits and climatic factors are closely related. Longitude and FPL, FSI, LDF show evidently positive correlation, however, longitude and BNSF, SFW show evidently negative correlation. Primarily, latitude and BLD, BHD, SFW, BNSF are evidently positively correlated, but latitude and the FSI and LDF show observably negative correlation. The annual rainfall (AR), annual mean temperature (AMT), and mean altitude (MA) have a greater impact on fruit characters.3.Lignans of S. sphenanthera in schisantherin A and deoxyschizandrin show highly positive correlation, while the deoxyschizandrin and y-schizandrin show highly negative correlation. The schisantherin A content of 85 germplasm (except 4 are not detected) is higher than the national pharmacopoeia standards, and higher levels of y-schizandrin have been found, but schisandrin (other than Gansu) content is very low.4. The contents of schisantherin A (average 0.724%) and deoxyschizandrin (an average of 0.812%) of Shangluo, Weinan, Henan and Ankang populations are high;the contents of y-schizandrin (an average of 1.085%) of Hanzhong, Baoji, Chongqing and Sichuan populations are high and average content of schisantherin A is 0.428%;Shanxi, in Zhejiang and Anhui populations, schisantherin A (average 0.310%),deoxyschizandrin (an average of 0.262%),and y-schizandrin (an average of 0.421%) content are at the middle level,intermediate between other populations. Schisandrin (mean 0.492%) and y-schizandrin (an average of 1.081%) is the highest in Gansu population.12 populations of S. sphenanthera based on four kinds of lignans Euclidean distance cluster into four categories.5.Eight pairs of selective primers of 85 germplasm in 12 populations are carried on AFLP to expand increases.In total,1384 locus are detected, and 1380 locus are polymorphic, percentage of polymorphic locus (P) is 99.711%.On average, each pair of primers is expanded by 173 locus. All germplasm have four commom bands with E-ACT/M-CTC-96.5,E-ACG/M-CAA-150.5, E-ACG/M-CAA-270.5,and E-ACG/M-CAC-90.5)6.62.35% of the S. sphenanthera germplasm are rich in specific locus, in total, there are 183. They are mainly in populations in Hanzhong of Shaanxi, Henan, Gansu, Chongqing, Zhejiang and Baoji of Shaanxi.A variety of groups with a specific band also indicate that there are genetic differences among populations.7.This study screens out four primer combinations (E-ACT/M-CTC, E-ACA/M-CTC, E-AGG/M-CTA, and E-ACA/M-CAA) amplify more specific bands, and the primer combinations can be applied to germplasm fingerprinting of S. sphenanthera.8.By AFLP markers Nei's genetic distance can devide 12 populations into five groups; Henan, Gansu, Sichuan and Shanxi together are a class. The second class contains Chongqing, Anhui and Baoji, and Weinan and Zhejiang respectively are two groups, besides, Hanzhong, Shangluo and Ankang cluster together.9. Analysis of molecular variance (AMOVA) reveals the genetic structure of populations to be abundant, especially in Gansu, Hanzhong, Shangluo and Henan population. The PhiST coefficient (ΦST) is 0.135,and it could further explain the genetic variation is mainly within populations, which is accord with phenotypic diversity analysis. As the gene exchanges among populations are subject to certain restrictions, in the protection and development of utilization, special attention should be paid to expand the selection of good single plant within populations.Through morphology, lignins contents and molecular marker studies, the major genetic variation of S. sphenanthera mainly exist within populations. Population variation in Shangluo, Hanzhong in Shaanxi, Henan and Gansu is notable. Thus, to do research of germplasm, the best choice is the population of rich genetic diversity, and to focus on the populations which vary greatly with the selection traits, such as FSI, LDF, FPL and BNSF. Secondly, we should expand the sample sizes within populations and increase the superior choice, and pay special attention to BLD, BHD, BFW, SFW. These researches in morphology, phytochemistry and molecular levels above reveal the genetic diversity of S. sphenanthera, and collect data for germplasm resources, which are significant for its rational development and use.