Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum

Abstract

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

The multipartite mitochondrial genome of Fallopia multiflora (Caryophyllales: Polygonaceae)

“…Fallopia multiflora (Hashuoh in Korean) is an important herb in Korea, Japan, and China (Han et al 2016). We extracted genomic DNA from fresh leaves collected from the Oriental Medicine Resource Center of Wonkwang University (geographic coordinate: N 35 56 0 38 00 , E 126 57 0 16 00 ).…”

Molecular markers based on chloroplast and nuclear ribosomal DNA regions which distinguish Korean-specific ecotypes of the medicinal plant Cudrania tricuspidata Bureau

“…HRM analysis is also a highly sensitive method for detecting SNPs, allowing sequence differences between species to be readily detected without the need for direct sequencing. ARMS-PCR and HRM analyses were recently used to discriminate among various medicinal plants, such as members of the diverse Panax genus and similar plant species Cynanchum wilfordii, C. auriculatum, and Polygonum multiflorum (Kim et al 2013;Han et al 2016). In the current study, we showed that SNPs in the MatK regions of plastid sequences and the ITS regions of nuclear sequences are effective, reliable tools for discriminating among C. tricuspidata ecotypes originating from the same country, such as Korea and China (Fig.…”

“…Most gene sequences are also highly conserved in various plant species, but considerable amounts of nucleotide variation have been identified in chloroplast intergenic spacer regions at the interspecies level and (rarely) at the intraspecies level (Wolfe et al 2004). In addition, nuclear ribosomal DNA internal transcribed spacer (ITS) sequences have recently been used to develop molecular markers to identify various medicinal plant species originating from Korea and China (Yang et al 2012;Han et al 2016). Hybrids may also be produced via cross-fertilization when similar species or ecotypes are cultivated in the same field.…”

“…Genetic markers based only on chloroplast intergenic sequences are likely to be insufficient for identifying specific species among hybrid (Jung et al 2014). Various DNA markers based on random polymorphic sequences have been used to classify similar medicinal plant species, including single nucleotide polymorphisms (SNPs) (Kim et al 2013;Han et al 2016). While the use of highly variable sequences from the plastid and nuclear genomes is important for barcoding, molecular markers to classify genetic diversity in C. tricuspidata have not yet been reported.…”

Practical application of the Bar-HRM technology for utilization with the differentiation of the origin of specific medicinal plant species

“…-Authentication of 11 collected samples of three different species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum (Han et al 2016).…”