DNA barcodes for the identification of Anoectochilus roxburghii and its adulterants

Chinese medicinal herbs have a similar appearance and are easily confused, complicating identification via traditional methods. This study provided a scientific approach, based on DNA barcoding, to accurately and rapidly identify Anoectochilus roxburghii and its adulterants. This technology complements traditional methods of identification of medicinal herbs. A comparison of the DNA barcodes matK, psbA-trnH and ITS2 was performed to verify that the ITS2 sequence is an effective marker for rapidly and accurately identifying A. roxburghii and its closely related species. Genomic DNA extracted from A. roxburghii and its adulterants were used as templates and the ITS2 sequence was amplified using PCR amplification and sequencing. Species identification was conducted using BLAST1 and neighbor-joining trees. The 12 samples were successfully classified into four species based on the ITS2 sequence. The ITS2 sequence length of A. roxburghii was 253 bp. The average intra-specific genetic distance of A. roxburghii was 0.0021, markedly lower than the inter-specific genetic distance between A. roxburghii and its adulterants (0.0380). Our findings illustrate that ITS2 sequence can accurately and efficiently distinguish A. roxburghii and its adulterants. In addition, the results provided reference for molecular identification of other Chinese herbal medicine.