Genetic diversity and hybridization of Pulsatilla tongkangensis based on the nrDNA ITS region sequence

Pulsatilla tongkangensis, a perennial medicinal herb growing on the cliffs of Tongkang, Kangwon Province, Korea, is accordingly named as P. tongkangensis as a new Pulsatilla species. Due to sharing similar morphological characters with other Pulsatilla species, how to accurately discriminate P. tongkangensis becomes a critical, urgently needed-solved problem. To understand the truth of P. tongkangensis derivation and the phylogenetic relationship in Pulsatilla, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) as the most frequently used marker for distinguishing plant species was amplified from P. tongkangensis in this study. According to sequence alignment result, three P. tongkangensis-specific nucleotide sites were obtained to vary from other Pulsatilla species, particularly one at 417 bp showing completely homology among P. tongkangensis accessions and completely dissimilarity between P. tongkangensis and other Pulsatilla species. As known fungal contamination is common in herbal medicinal materials when improperly processed and stored, using universal primers of the ITS region could not only plant DNA but parasitic fungal DNA. Thus, to improve the amplification condition of the ITS region from P. tongkangensis and design plant-specific primer would the following focus of our study. This work will be useful for clearly understanding the phylogenetic relationship between P. tongkangensis and other species belonging to the genus Pulsatilla.