Authentication of Rubus coreanus and Related Species via DNA Barcoding and HPLC Approaches

Background: Rubus coreanus and R. occidentalis are commonly sold as, ‘Bokbunja’, in the Korean market. However, according to Korean Pharmacopoeia, ‘Bokbunja’ refers to the unripened fruits of R. coreanus. Therefore, authentication of R. coreanus and its related species is necessary to prevent adulteration and improper use. Methods and Results: Genomic DNA was extracted from leaves, stem and fruits of R. coreanus, fruits of R. occidentalis and leaves of R. longisepalus and R. hirsutus. The anthocyanin profile of R. coreanus and R. occidentalis was identified by high performance liquid chromatography/diode-array detection. We designed four universal barcoding primer sets with optimal restriction enzyme sites to identify polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) between R. coreanus, the newly assembled chloroplast genome of R. occidentalis, and two related species. In addition, two Insertion or Deletion (InDel) markers were also developed. These markers were successfully used to distinguish each species in nine samples. Conclusions: In this study, we sequenced and assembled the complete chloroplast genome of R. occidentalis and developed a DNA barcoding method to differentiate it from other related species. Thus, anthocyanin profiling, PCR-RFLPs, and InDel markers are useful authentication tools to distinguish R. coreanus from R. occidentalis.