Development of species-specific chloroplast markers for the authentication of Gynostemma pentaphyllum and their distribution in the Korean peninsula.

Gynostemma pentaphyllum is a traditional oriental medicinal herb used as tea since ancient time. Among Gynostemma species, G. pentaphyllum has more active chemical components and better therapeutic effect. It is used to cure depression, diabetes, anxiety, hyperlipidemia, fatigue, immunity, cancer, and oxidative stress. Overexploitation of G. pentaphyllum for its medicinal benefits has been on a rise, due to which they are adulterated or mistakenly identified with other members of Gynostemma species. Hence, we used chloroplast universal regions such as ycf3, accD, petD, psbB and their polymorphism to distinguish G. pentaphyllum from other Gynostemma species. By using the species-specific primers derived from the above regions, we established a multiplex allele-specific PCR for the authentication of G. pentaphyllum from other species. Thus the PCR reaction produced unique amplicons of size 244 bp and 438 bp for G. pentaphyllum amplified by the primers flanking ycf3, and accD regions respectively. While a 607 bp, and 787 bp amplicons from the primers targeting psbB, and petD regions distinguished G. longipes, G. burmanicum, and G. pubescens species. Moreover, these primers were successful to analyze the dried tea samples of Gynostemma as well. Thus, the developed molecular markers could authenticate different Gynostemma species as well as its products thereby preventing the mistaken-identity of this medicinal herb.

Molecular authentication of Gynostemma pentaphyllum through development and application of random amplification polymorphic DNA sequence-characterized amplified region marker.

Due to the morphological similarities of aerial parts, it is difficult to distinguish Gynostemma pentaphyllum from Cayratia japonica, which is usually an adulterant of the former. To develop a reliable method for the identification and authentication of G. pentaphyllum, a combination of random amplification polymorphic DNA (RAPD) technique with sequence-characterized amplified region (SCAR) markers was studied. Twenty-five samples of G. pentaphyllum and two samples of C. japonica were collected from different regions in Guangxi or bought from different provinces in China. Through the RAPD analysis, significant genetic polymorphism was observed among the intraspecies samples of G. pentaphyllum. Furthermore, a specific marker, J-750, was obtained for authentication. Therefore, the SCAR marker for G. pentaphyllum (359 bp) was developed from the RAPD amplicon. With PCR amplification using the SCAR primers, a specific band of 359 bp was distinctly visible for all tested samples of G. pentaphyllum, but was absent in the samples of C. japonica. Furthermore, the results revealed that the SCAR marker was useful for the identification and authentication of G. pentaphyllum irrespective of whether samples were fresh, dry, or of commercial origin. The SCAR marker obtained in this study successfully authenticated G. pentaphyllum through an integrated PCR system containing SCAR and control primer combinations of two pairs. In addition, it was also used for simultaneous discrimination of G. pentaphyllum from C. japonica.

Characterization of Global Transcriptome Using Illumina Paired-End Sequencing and Development of EST-SSR Markers in Two Species of Gynostemma (Cucurbitaceae).

Gynostemma pentaphyllum is an important medicinal herb of the Cucurbitaceae family, but limited genomic data have hindered genetic studies. In this study, transcriptomes of two closely-related Gynostemma species, Gynostemma cardiospermum and G. pentaphyllum, were sequenced using Illumina paired-end sequencing technology. A total of 71,607 nonredundant unigenes were assembled. Of these unigenes, 60.45% (43,288) were annotated based on sequence similarity search with known proteins. A total of 11,059 unigenes were identified in the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. A total of 3891 simple sequence repeats (SSRs) were detected in 3526 nonredundant unigenes, 2596 primer pairs were designed and 360 of them were randomly selected for validation. Of these, 268 primer pairs yielded clear products among six G. pentaphyllum samples. Thirty polymorphic SSR markers were used to test polymorphism and transferability in Gynostemma. Finally, 15 SSR makers that amplified in all 12 Gynostemma species were used to assess genetic diversity. Our results generated a comprehensive sequence resource for Gynostemma research.

[Analysis phylogenetic relationship of Gynostemma (Cucurbitaceae)].

The sequences of ITS, matK, rbcL and psbA-trnH of 9 Gynostemma species or variety including 38 samples were compared and analyzed by molecular phylogeny method. Hemsleya macrosperma was designated as outgroup. The MP and NJ phylogenetic tree of Gynostemma was built based on ITS sequence, the results of PAUP phylogenetic analysis showed the following results: (1) The eight individuals of G. pentaphyllum var. pentaphyllum were not supported as monophyletic in the strict consensus trees and NJ trees. (2) It is suspected whether G. longipes and G. laxum should be classified as the independent species. (3)The classification of subgenus units of Gynostemma plants is supported.

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

OBJECTIVE: To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. METHODS: Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. RESULTS: There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. CONCLUSION: RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

Polyploid origins in Gynostemma pentaphyllum (Cucurbitaceae) inferred from multiple gene sequences.

The genus Gynostemma (Cucurbitaceae) constitutes a polyploid group of perennial creeping herbs, in whose evolution polyploidization is a key component. With the largest variety of cytotypes (2n=22, 44, 66 and 88) in Gynostemma, G. pentaphyllum is also the most widespread species in this genus. In the present study, we inferred the origins of polyploids in G. pentaphyllum using sequences of the plastid intergenic spacers (trnL-trnF, psbB-psbF and rpl20-rps12) and cloned DNA sequences from two nuclear regions (RPB2 and nrDNA ITS). Phylogenetic analyses of the separate and the combined nuclear gene datasets all supported autoploid origins of polyploids in G. pentaphyllum. Three polyploid populations were more closely related, indicating that significant genetic differentiation may have occurred between diploids and polyploids. We concluded that polyploidization might be an important evolutionary mechanism in the diversification of G. pentaphyllum. On the other hand, no chloroplast DNA (cpDNA) variation was detected in ingroups except the octoploid DL 8x, which possessed a different cpDNA haplotype from the other populations of G. pentaphyllum. This can be explained by limited sample sizes, possible extinction of its diploid progenitors and/or the occurrence of chloroplast transfer through hybridization with other Gynostemma species. However, the distribution of cytotypes in G. pentaphyllum was not as typical as many other autopolyploid complexes. Polyploidization failed to contribute significantly to the expansion of its geographic range. The geographic distribution of diploids and polyploids in G. pentaphyllum may be associated with the past ecological environments of different areas, especially during the glacial period.

[Molecular characters of Centella asiatica found with RAPD technology].

OBJECTIVE: To analyze the DNA molecular characters of Centella asiatica with RAPD technology. METHODS: With the genomic DNA as templates extracted from various source of Centella asiatica samples, optimized RAPD PCR reaction systems had been used. The random promers had been screened to amplify the specific molecular fragments of Centella asiatica. RESULTS: The specific genetic bands of Centella asiatica species from various habitats were established which were highly stable and repeatable and obviously different from those of other families, genuses of plants such as Gynostemma pentaphylum, Tobacco, Cayratia japonica. CONCLUSION: The developed method of RAPD analysis for the genetic character bands of Centella asiatica could be applied to identify real Centella asiatica from its spurious breed plants. The genetic character bands of Centella asiatica amplified with the RAPD method show high homogeneous in several samples from different habitats.

[Identification of Gynostemma from their adulterant Cayratia japonica by PCR-RFLP].

OBJECTIVE: To establish a convenient and effective method for the identification of Gynostemma and Cayratia japonica. METHOD: Eight species, including Gynostemm pentaphyllum, G. pentagynum, G. cardiospermum, G. longipe, G. yixingense, G. laxiflorum, G. guangxiense and C. japonica were investigated through PCR - RFLP of six chloroplast DNA fragments. The six gene fragments were digested by six restriction endonuclease respectively, including Taq I, Hpa II, EcoR I, Rsa I, Hha I, Hind III. RESULT: Seven species of Gynostemma and their adulterant could be identified by trnK1f-trnK2r and Rsa. CONCLUSION: PCR - RFLP provides a quick, reliable molecular marker technique for identification of Cynostemma and their adulterant Cayratia japonica.

[Species and distribution of the medicinal plants peculiar to Guizhou].

OBJECTIVE: To review the species and distribution of the medicinal plants peculiar to Guizhou and provide evidence for application, protection and collection. METHOD: Open-air investigation, data collection and specimen identification. RESULT: More than eighty kinds of the medical plants peculiar to Guizhou have been identified. CONCLUSION: Guizhou has a diversity of medicinal plants. The area of distribution of most species is restricted and the population is small. Some of the species have higher medicinal and scientific research values.

[Comparison of total saponin content in three wild species of Gynostemma on Mount Emei].

Analysed by TLC and spectrometry, the content of the total saponin in three wild species of Gynostemma on Mount Emei is found to be the highest in July and to be the lowest in the seedling stage. Among the three, the content of the total saponin in Gynostemma pentaphyllum is the highest, that in Gynostemma pubescens lower and that in Gynostemma longipes the lowest.