ABSTRACT
Objective:To establish a molecular identification method for Bupleurum chinense seeds based on ribosomal DNA internal transcribed spacer (ITS) sequence, ensuring the species authenticity of the cultivated seeds of B. chinense. Method:A total of 59 seeds samples of B. chinense and its main cultivated species, marketed B. chinense were collected. The effect of different sampling amounts and different water bath conditions on DNA extraction quality of the seeds was investigated, a DNA extraction method for seeds of Bupleurum was established. Their ITS sequences were obtained by polymerase chain reaction (PCR) and bidirectional sequencing. In addition, 34 ITS sequences of main cultivated Bupleurum species, such as B. chinense, B. scorzonerifolium, B. falcatum and B. smithii, were downloaded from GenBank to enrich identification database of B. chinense seeds. The neighbor-joining (NJ) dendrogram were constructed by MEGA-X 10.0.5 software to investigate the the species identification ability of ITS sequences for B. chinense seeds. And DNA barcoding identification of marketed B. chinense seeds was conducted based on BLAST method and NJ dendrogram method. Result:In total, 59 ITS sequences were obtained. ITS sequences of B. chinense could be divided into six haplotypes, including seven variable sites. The NJ dendrogram indicated that all the haplotypes of B. chinense could form independent branches, which could be distinguished from other cultivated species of Bupleurum in the collected samples, and possessed the ability to identify species of B. chinense seeds. Based on ITS sequence barcoding identification, 3 of the 19 marketed B. chinense seeds were B. falcatum with a counterfeit rate of 15.8%. Conclusion:DNA barcoding technology based on ITS sequence can accurately and reliably identify B. chinense seeds and its adulterants, providing reference for the standardization construction of Chinese medicinal materials seeds.