Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences.

This study was performed to identify and analyze the phylogenetic relationship among four herbaceous species of the genus Paeonia, P. lactiflora, P. japonica, P. veitchii, and P. suffruticosa, using DNA barcodes. These four species, which are commonly used in traditional medicine as Paeoniae Radix and Moutan Radicis Cortex, are pharmaceutically defined in different ways in the national pharmacopoeias in Korea, Japan, and China. To authenticate the different species used in these medicines, we evaluated rDNA-internal transcribed spacers (ITS), matK and rbcL regions, which provide information capable of effectively distinguishing each species from one another. Seventeen samples were collected from different geographic regions in Korea and China, and DNA barcode regions were amplified using universal primers. Comparative analyses of these DNA barcode sequences revealed species-specific nucleotide sequences capable of discriminating the four Paeonia species. Among the entire sequences of three barcodes, marker nucleotides were identified at three positions in P. lactiflora, eleven in P. japonica, five in P. veitchii, and 25 in P. suffruticosa. Phylogenetic analyses also revealed four distinct clusters showing homogeneous clades with high resolution at the species level. The results demonstrate that the analysis of these three DNA barcode sequences is a reliable method for identifying the four Paeonia species and can be used to authenticate Paeoniae Radix and Moutan Radicis Cortex at the species level. Furthermore, based on the assessment of amplicon sizes, inter/intra-specific distances, marker nucleotides, and phylogenetic analysis, rDNA-ITS was the most suitable DNA barcode for identification of these species.

Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials.