Chloroplast DNA-derived markers for the authentication of oriental medicinal Rubus species and mistaken identity of bokbunja in the local markets of Korea

Abstract

The traditional oriental medicine bokbunja, prepared from immature berries of Rubus coreanus is used as an anti-oxidant, diuretic, and cure for impotence. The bokbunja wine made from fermented fruits of bokbunja has been used as a functional food as well. However, the usage of bokbunja has been problematic over the years due to the abundance of mistakenly identified berries such as Rubus chingii, Rubus crataegifolius, and Rubus occidentalis. Thus, here we developed a method for the molecular differentiation of Rubus species as well as the authentication bokbunja from other Rubus species. We screened several sequences from the chloroplast DNA of these species and found that the rpl16 region was polymorphic for R. coreanus and R. occidentalis, while the trnG–trnS intergenic spacer region was polymorphic for R. chingii and R. crataegifolius. Species-specific primers were designed and a multiplex PCR was performed by combining the markers at the rpl16 and trnG–trnS regions. Amplicons of 686 bp for R. coreanus and 478 bp for R. occidentalis were produced by the primers 5′ Rcor or 5′ Rocci, respectively, with 3′ rpl16; whereas, amplicons of 389 bp for Rubus crataegifolius and 180 bp for R. chingii were produced by 5′ Rcra or 5′ trnG–trnS, respectively, and 3′ Rcra/Rchi. The deduced molecular markers were utilized to authenticate the bokbunja products and demonstrated that the majority of bokbunja samples from the markets were adulterant berries. Hence, our results indicate that the produced molecular markers can serve as an effective tool to authenticate bokbunja.