Intraspecific discrimination of Paris polyphylla in Wudang Mountain area based on ITS2 / 中草药

ABSTRACT

Objective: DNA barcoding technology, a molecular identification method, is applied to Paris polyphylla collected from Wudang Mountain area in order to solve the differential problem caused by small morphology difference, make authentic origins canonical, and guarantee the quality and clinical curative effect of this medical material in our area. Methods: In this study, the second internal transcribed spacers (ITS2) of ribosomal DNA were amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. To perform a phylogenetic study, the genetic distances were computed using molecular evolutionary genetics analysis 6.0 (MEGA 6.0) in accordance with the Kimura-2-parameter (K2P) model. The phylogenetic tree was constructed using the neighbor-joining (NJ) and Kimura-2-parameter methods. The ITS2 secondary structures were predicted using ITS2 database and website which was found by Keller. Results: Identified by BLAST, P. polyphylla collected in Wudang mountain area was proved to be precise. The results showed that the ITS2 sequence length was 232 bp. It was indicated that their K2P distance were 0-0.073 6 and the average was 0.027 2. Compared with the gene sequence of P. polyphylla, there were 16 variation loci in P. fargesii and only one variation locus in P. polyphylla var. chinensis. As seen from the NJ tree, P. polyphylla in this area was mainly divided into two categories, P. polyphylla and P. fargesii. Obviously, P. polyphylla var. chinensis was a variation of P. polyphylla. Meanwhile, it was not tenable that P. polyphylla var. stenophylla and P. polyphylla var. latifolia were the variations of P. polyphylla because they belonged to a branch. To compare the ITS2 secondary structure of P. polyphylla, we noticed the differences in the amount, size, and position of loop of helix zone. Conclusion: DNA barcoding technology opens up a new way for the identification of P. polyphylla. Regardless of what methods were used, such as similarity search, nearest distance, and NJ tree method, the analysis results have fully demonstrated that ITS2 sequences could be used to correctly identify the different varieties of P. polyphylla. It is identical in 232 base sequence of ITS2 to P. polyphylla var. stenophylla, P. polyphylla var. latifolia, and P. polyphylla. The results computed by Mega 6.0 also show that they belong to a branch. It is more appropriate to say P. polyphylla var. stenophylla and P. polyphylla var. latifolia are variants of P. polyphylla. Hence, as a DNA barcode sequence, ITS2 can be applied to intraspecific discrimination of P. polyphylla. Furthermore, the application of ITS2 in the identification of traditional Chinese medicine has an important prospect. And it can be used to correct identification of germplasm resources in GAP procedures.