Genetic Diversity Of Three Species Of Tupistra Ker-gawl. In Qinling-bashan Mountain Area

Tupistra Ker-Gawl，a genus of family Liliaceae, is a perennial herb mainly distributedin Asia. There are20species and12varieties in this genus grow in China. It is a group ofplants which have high economic value，has been used in traditional Chinese medicine.Steroidal saponins, sapogenin and other various bioactive constituents were isolated inTupistra Ker-Gawl. Habitat destruction and overexploitation leads to threatened. In thisstudy, aiming to evaluate evllutionary potential and propose conservational stratagem, datafrom16taxa of Qinling-Bashan Mountain areas were used to measure genetic diversityand genetic structure within three species.The high-quality and high-purity total genome DNA from dried leave of T. chinensisBaker was isolated using CTAB method. The concentration of Mg2+, dNTP, primer andTaqDNA polymerase, which greatly influence ISSR-PCR reaction, were optimized. Theoptimized reaction condition was established as follows: the PCR was performed in a10μLvolume system, which contains1×buffer,1U TaqDNA polymerase,2mmol/L MgCl2,0.2mmol/L dNTP,0.75μL primer.14ISSR primers with stable amplification and richpolymorphism were screened from80primers. The optimized annealing temperature foreach primer in ISSR-PCR reaction was proposed. This study will be helpful to research onthe genetic diversity analysis and germplasm identification of T. chinensis Baker.In the study, ISSR (inter-simple sequence repeat) polymorphism was used todetermine genetic diversity in T. chinensis, T. wattii and T. delavayi. Based on these results,high genetic diversity in species lever (PPL=79.04%, He=0.2664, I=0.3975) werefound among populations in Tupistra chinensis, while genetic diversity at population levelwere high (PPL=40.00%, He=0.1492, I=0.2209), T. chinensis performed the highestgenetic diversity among the three sepecies. Analysis of molecular variance (AMOVA)showed a strong genetic differerntiation happened among populations (GST=0.4282; FST=0.4170). Gene flow among populations was limited with the value of0.6676. Cluster andprincipal component analyses data clearly separated T. chinensis, T. wattii and T. delavayi. using the UPGMA cluster analysis and the PCA analysis Within the T. chinensis group, thegenetic distance of ten taxa between Qinling and Bashan Mountain areas was far,illustrating the relatively high genetic diversity present in different habitats. Mating system,clone growth and genetic drift jointly constitute the genetic structure of T. chinensis. Theresults of this study suggest that appropriate in situ conservation interventions andstrategies may have important implications for the conservation of Tupistra Ker-Gawl.