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

Abstract

ABSTRACT. 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.

Peer Review #2 of "Assessing universality of DNA barcoding in geographically isolated selected desert medicinal species of Fabaceae and Poaceae (v0.1)"

“…Phylogenetic analysis provided a better species resolution than the nucleotide analysis (Clement and Donoghue 2012;Kim et al, 2016) and has shown that despite of the fact that all of the PeerJ reviewing PDF | (2017:11:21621:1:1:NEW 7 Feb 2018)…”

Peer Review #1 of "Morphological and molecular barcode analysis of the medicinal tree Mimusops coriacea (A.DC.) Miq. collected in Ecuador (v0.1)"

“…Plant Working Group 2009). Although, the two proposed loci for barcodes are from plastid genome and includes the rbcL and matK (Techen et al, 2014), other loci including ITS1 and ITS2 are widely used for medicinal plants (Kim et al, 2016). Furthermore, the ITS2 region is suggested as a barcode for species identification over rbcL and matk (Zhang et al, 2016).…”

Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers

“…The nrDNA-ITS regions were amplified using the primers ITS1 (5 -TCC GTA GGT GAA CCT GCG G-3 ) and ITS4 (5 -TCC TCC GCT TAT TGA TAT GC-3 ) with previously-reported PCR parameters [27,28]. PCR reactions were performed in 50 µL mixtures consisting of 10 mM Tris-HCl (pH 9.0), 2.5 mM MgCl 2 , 200 µM of each dNTP, 10 mM (NH 4 ) 2 SO 4 , 0.5 U Taq DNA polymerase (SolGent, Daejeon, Korea), 0.5 µM of each primer, and 15 ng of template DNA.…”