ABSTRACT
Objective: To discriminate traditional She medicine Gegongniugen (the root of Rubus chingii Hu) from its confusable species of genus Rubus based on ITS2 barcode, and to guarantee the medicine quality and clinical effect of Gegongniugen. Methods: A total of 140 samples of R. corchorifolius L. f., R. hirsutus Thunb., R. parvifolius L., R. coreanus Miq., R. buergeri Miq., R.tsangorum Hand. -Mazz., R. trianthus Focke, R. jiangxiensis Z. X. Yu, W. T. Ji et H. Zheng were collected from Zhejiang, Guangxi, Jiangxi, and Anhui provinces. Genomic DNA was extracted from the collected samples, followed by ITS2 sequences amplification through PCR, bi-directional sequencing, assembly and annotation based on HMMer method. At the same time, 37 other sequences of corresponding species were downloaded from GenBank. Sequence characteristics such as lengths, GC contents, and variable sites were analyzed by Gene Tool software while genetic distance calculation and build-up of neighbor-joining (NJ) phylogenetic tree were carried out through Clustal X and MEGA 7.0. Results: The lengths of ITS2 sequences of Gegongniugen and its nine confusable species of genus Rubus ranged from 211 to 213 bp with GC content of 52.1%-58.0%; These ITS2 sequences had 14 mutation sites and one insertion/deletion site; The average intra-specific K2P genetic distance of Gegongniugen was 0.007 2, which was significantly lower than the respective inter-specific K2P genetic distance between Gegongniugen and its confusable species of genus Rubus. The genetic distantes between Gegongniugen and R. corchorifolius or Gegongniugen and R. jiangxiensis were minimum, while that between Gegongniugen and R. hirsutus was maxmum. It was obvious in the N-J phylogenetic tree that the ITS2 sequences of Gegongniugen formed into one separate clade, which could be successfully distinguished other nine species. Conclusion: ITS2 sequence can be used as an effective DNA marker to discriminate the original plants of Gegongniugen and its confusable species of genus Rubus, which provided an important molecular evidence for the identification of traditional She medicines and contributed to the information supervision of traditional She medicine market.