Development of a molecular marker to discriminate Korean Rubus species medicinal plants based on the nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL-F intergenic region sequences

Abstract

Rubi Fructus (Bokbunja in Korean) is a traditional oriental medicine often prescribed as the immature fruit of Rubus coreanus in the Korean pharmacopoeia or Rubus chingii in the Chinese pharmacopoeia. However, Rubus crataegifolius is presently the most popular form in the market. Correctly identifying these fruits is difficult due to morphological similarities in their dried immature forms. Therefore, nucleotide sequences of nuclear ribosomal DNA (nrDNA), internal transcribed spacer, chloroplast DNA (cpDNA) trnL-F, and psbA-trnH intergenic spacer were analyzed to develop objective methods for discrimination among R. coreanus, R. crataegifolius, and R. chingii. Based on trnL-F intergenic sequences, three sequence characterized amplified region (SCAR) markers (CorF/CorR, HwaF2/HwaR2, SanF/SanR) were developed to distinguish the three species. Furthermore, phylogenetic analysis of the trnL-F intergenic sequences showed that R. crataegifolius is widely distributed in the Korean Rubi Fructus market as Korean product and imported goods from China. Single nucleotide polymorphism (SNP) that can be used to discriminate Korean Bokbunja medicine from those imported from China was developed, established the SNP marker, and conducted real-time PCR with a designed inflorescence probe. The developed SCAR marker and SNP probe were useful for distinguishing and authenticating the Korean and Chinese species that constitute Rubi Fructus medicines.