Study On DNA Barcode Identification Of Codonopsis Pilosula, Acacia Flower (Phi), Wujiapi Herbs And Their Mixed Products

DNA barcoding is a hot research technique which uses short DNA sequences to identify species. A DNA barcoding system for herbal medicine based on ITS2, as the core DNA barcode, has been established. And an online DNA barcoding database has been constructed. The principle for DNA barcoding traditional Chinese medicine has been added to supplement 3 of the Chinese Pharmacopoeia (2010 edition). DNA Barcoding provides a powerful new tool for identifying traditional Chinese medicine and solving problems about the substitutes and adulterants.(1) The ITS/ITS2 barcodes were used to simply and effectively identify Codonopsis Radix and its adulterants. In this study, ITS regions were amplified using PCR from fifty-eight samples of Codonopsis Radix and ITS2 regions were obtained from the ITS sequences using the hidden Markov model (HMMer)-based annotation methods. Species identification efficiency of ITS/ITS2 sequences were evaluated using BLAST1 and nearest distance methods. The results indicated that the sequences lengths of ITS regions of Codonopsis Radix were 654-655 bp, and the lengths of ITS2 regions were 239 bp. The maximum intraspecific genetic distances among Codonopsis Radix were obviously lower than the minimum interspecific genetic distance between Codonopsis Radix and its adulterants. Therefore, ITS/ITS2 regions can stably and accurately distinguish Codonopsis Radix and its adulterants.(2) The ITS2 barcode was used to accurately identify Albiziae Cortex, Albiziae Flos and their adulterants. In total,49 samples from Albiziae Cortex, Albiziae Flos and their adulterants were collected. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to evaluate the identification efficiency of the ITS2 barcode. Our conclusion reveals that the intraspecific genetic distances of Albizia julibrissin are lower than the interspecific genetic distances between A. julibrissin and its adulterants. The NJ-tree showed that A. julibrissin and their adulterants can be easily differentiated according to their monophyly. The ITS2 barcode is appropriate to identify Albiziae Cortex, Albiziae Flos and their adulterants.(3) The ITS2 barcode was used to identify 69 samples belonging to six species, including Acanthopanacis Cortex and its adulterants. The nearest distance, SNPs, and neighbor-joining tree methods were used to evaluate the identification ability of the ITS2 barcode. According to the K2P model, the intraspecific distance of Eleutherococcus nodiflorus ITS2 sequences ranged from 0 to 0.0132. The minimum interspecific distance between E. nodiflorus and E. giraldii was 0.0221, which is larger than the maximum intraspecific distance of E. nodiflorus. Three stable SNPs in ITS2 can be used to distinguish Acanthopanacis Cortex and its closely related species. The NJ tree indicated that Acanthopanacis Cortex samples clustered into one clade, which can be distinguished clearly from its adulterants. Moreover, secondary structure of ITS2 provides another dimensionality to identify species. Our results concluded that the ITS2 barcode is effective to identify Acanthopanacis Cortex, and DNA barcoding is a convenient tool for medicine market supervision.