Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BACKGROUND: While DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng). METHODS: Genomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke's Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA-trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0. RESULTS: The accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA-trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA-trnH was higher than the corresponding intraspecific distance. Additionally, psbA-trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method. CONCLUSION: While psbA-trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA-trnH was a more suitable barcode for authentication of Uncaria macrophylla.

[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.

[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.