畲药搁公扭根基原植物及其同属易混种的ITS2条形码鉴别

目的基于ITS2条形码序列对畲药搁公扭根及其同属易混种进行分子鉴定,以保证药材使用的正确性和临床疗效。方法分别于浙江、广西、江西和安徽等地采集搁公扭根基原植物掌叶覆盆子及其9种同属易混种高粱泡、山莓、蓬蘽、茅莓、插田泡、寒莓、东南悬钩子、三花悬钩子和武夷悬钩子样品共140份。对采集样品进行基因组DNA提取、ITS2基因片段PCR扩增并双向测序、拼接获得相应序列,基于HMMer方法注释获取ITS2序列,同时下载Gen Bank相应物种ITS2序列37条,使用Gene Tool软件分析ITS2序列长度、GC量、变异位点等情况,利用Clustal X和MEGA7.0软件计算种内、种间遗传距离,构建邻接(NJ)系统发育树。结果搁公扭根及其9种同属易混种ITS2序列长度为211~213 bp,GC量为52.1%~58.0%;搁公扭根基原植物与其他物种共有14个变异位点,一处插入/缺失;搁公扭根种内平均K2P遗传距离为0.007 2,显著小于其他9个易混种,其中与山莓和武夷悬钩子进化距离最近,与蓬蘽最远;NJ进化树中搁公扭根各单倍型序列单独聚为一支,可与其他物种成功区分。结论 ITS2序列能够成功鉴别搁公扭根基原植物及其9种同属易混种,为畲药DNA条形码分子鉴别提供了有力佐证,助力畲药流通信息监管。

Identification of traditional She medicine Gegongniugen and its confusable species of genus Rubus using ITS2 barcode

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.