应用ITS2条形码及种子形态鉴定柴胡属种子

目的:采用传统形态鉴定方法结合DNA条形码分子检测技术准确鉴定柴胡属种子。方法:收集41份市售柴胡属种子和GenBank数据库中下载15个品种75条核糖体DNA第二内部转录间隔区(ITS2)序列。利用体视显微镜和游标卡尺进行种子外观形态特征的观察和记录,测定千粒重。提取种子的基因组DNA,采用聚合酶链式反应(PCR)扩增,双向测序得ITS2序列。基于BLAST法,邻接(NJ)系统进化树法,Kimura双参数模型(K2P)遗传距离法和ITS2序列二级结构进行物种鉴定。结果:不同基原的柴胡属种子在长、宽、横切面及千粒重等方面存在细微的差别;柴胡种子的ITS2序列有2个种内变异位点,3种单倍型,种内最大遗传距离0.009小于种间最小遗传距离0.032;NJ系统进化树中柴胡和狭叶柴胡分别聚为独立一支,具有良好的单系性;ITS2序列二级结构可以弥补NJ树在变种鉴定方面的不足。41份柴胡属种子样品中有30份柴胡,3份狭叶柴胡,5份三岛柴胡,2份藏柴胡及1份小叶黑柴胡。结论:市售柴胡种子存在来源多样、基原混乱的问题。基于ITS2条形码和种子形态鉴定相结合的方法可以准确鉴定柴胡属种子,为制定柴胡种子质量标准,规范柴胡种植,从源头解决柴胡药材质量问题提供科学依据,并为其他药用植物种子或者种子类药材的准确鉴定提供参考。

Identification of Bupleurum Seeds by ITS2 Genetic Barcoding and Seed Morphology

Objective To accurately identify Bupleurum seeds by traditional morphological identification method combined with DNA barcoding technique.Method A total of 41 seed samples on the market were collected and 75 ribosomal DNA internal transcribed spacer 2 (ITS2) sequences of 15 varieties were downloaded from the GenBank database as experimental materials. The seeds were measured and observed by stereomicroscope and vernier caliper, and their 1 000-grain weights were calculated. Genomic DNA was extracted from the seeds and used as a template, and ITS2 sequences were amplified using polymerase chain reaction (PCR) and bidirectional sequencing. Species identification was conducted based on BLAST method, neighbor-joining (NJ) phylogenetic tree method, Kimura two-parameter model (K2P) genetic distance method, and secondary structure of ITS2 sequence.Result There were slight differences in the length, width, cross-section, and 1 000-grain weight among Bupleurum seeds from different origins. The ITS2 sequences of B. chinense seeds had 2 intraspecific variable sites and 3 haplotypes, the maximum intraspecific genetic distance (0.009) was far smaller than the minimum interspecific genetic distance (0.032). B. chinense and B. scorzonerifolium in the NJ phylogenetic tree were clustered into independent branches with good monophyletic property. The secondary structure of ITS2 sequences could make up for the shortcomings of NJ tree in identifying variants. The collected 41 seeds included 30 B. chinense seeds, 3 B. scorzonerifolium seeds, 5 B. falcatum seeds, 2 B. marginatum var. stenophyllum seeds, and 1 B. smithii var. parvifolium seeds.Conclusion The B. chinense seeds on the market have problems of diverse sources and chaotic origins. Based on the combination of ITS2 gentic barcoding and seed morphological identification, the Bupleurum seeds can be accurately identified, which provides scientific bases for establishing the quality standard of Bupleurum seeds, standardizing the cultivation of B. chinense, and solving the quality problems of B. chinense from the source, and provides a reference for the accurate identification of other medicinal plant seeds or seed medicinal materials.