Analysis For Genetic Diversity And Establishment Of Authentication Methods On Molecular Level For Medicinal Plant Gynostemma Pentaphyllum From Guangxi

Gynostemma pentaphyllum, which belongs to Cucurbitaceae,Gynostemma BL.Herbs, is the one of special medicinal plants in Guangxi. More than 100 kinds of saponin substances were isolated and identified from Gynostemma pentaphyllum, including eight kinds of saponin whose structure were the same with ginsenoside. It is verified that Gynostemma pentaphyllum is the one of minority plants containing ginsenoside except Araliaceae plants, possessing the considerable pharmaceutical and commerce values. However, its development and utilization are restricted due to its chaotic genetic background and confusable with Cayratia japonica.It is the major aim for the study to screen the appropriate RAPD random primers, to analyze the genetic diversity of Gynostemma pentaphyllum in different regions of Guangxi and to construct DNA fingerprints with random amplified polymorphic DNA (RAPD) technique,which is beneficial for breeding and germplasm resources protection.On the other hand, we attempt to adopt DNA markers technique to authenticate of Gynostemma pentaphyllum and establish authentication method on molecular level for medicinal plant Gynostemma pentaphyllum species .Total genomic DNA of Gynostemma pentaphyllum and Cayratia japonica were successfully extracted from fresh and dry samples, applying modified CTAB protocols. Twenty decamer oligonucleotides were selected out from thousands of random primers through bioinformatics methods and verified by the RAPD-PCR optimized reaction system. Nineteen of them could amplify Gynostemma pentaphyllum genomic DNA effectively. The obtaining RAPD fingerprints were clear and reproducible. A total of 354 RAPD bands generated from the nineteen primers, including 294(83.3%) polymorphic bands. The molecular weight of the amplified fragments ranged from 200bp to 5000bp. NTSYS-pc software were used to compute genetic similarity (GS) and a dendrogram was constructed based on GS using UPGMA method and Tree plot program. The genetic similarity of Gynostemma pentaphyllum was in the range of 0.5367~0.7825 (Average=0.6353). The dendogram reflected that 8 samples clustered into two taxa,and it was in accordance with their geographic origin and growing environment.Based on the analysis of Gynostemma pentaphyllum DNA fingerprints, we obtained two RAPD markers (length1≈500bp, length2≈750bp), named J-500and J-750, which could be used for authentication of Gynostemma pentaphyllum. The RAPD markers J-750 were cloned, sequenced and analyzed. The bioinformatics analysis revealed that the sequence similarity of J-750 were more than 97% and these eight DNA sequences were detected for the first time. According to the sequence, we adopted Oligo 6 software to design specific primers and made RAPD markers convert into SCAR markers. Utilizing the SCAR marker and 18SrRNA gene sequences through PCR technique, we could identify the Gynostemma pentaphyllum and Cayratia japonica accurately. In this study, the nineteen random primers selected out by bioinformatics methods were appropriate for genetic diversity analysis of Gynostemma pentaphyllum, whose genetic background is complex. The result of authentication shows that it is a rapid and effective method we established to identify Gynostemma pentaphyllum. This study provides some references to develop a new molecular authentication method in different medicinal plants.