藏药兔耳草常用基原的ITS2序列鉴定研究

目的 利用DNA条形码内转录间隔区2（internal transcribed spacer 2，ITS2）序列针对自采及流通使用环节的不同基原兔耳草进行分析，建立一种快速鉴定常用兔耳草的方法，并分析市场流通的主流品种。方法 采用试剂盒提取101份兔耳草样品的DNA，针对其ITS2片段进行PCR扩增并双向测序，获取ITS2序列信息；并从GenBank上下载相关序列28条。运用MEGA6.0对129条ITS2序列进行比对分析，计算种内和种间K2P遗传距离，构建邻接法（neighbor-joining，NJ）系统进化树，并预测ITS2二级结构。采用BLAST法及NJ聚类分析法分析市场流通兔耳草基原。结果 除四川部分区域所产全缘兔耳草Lagotis integra与革叶兔耳草L.alutacea种间亲缘关系近外，短筒兔耳草L.brevituba、全缘兔耳草、圆穗兔耳草L.ramalana、革叶兔耳草、短穗兔耳草L.brachystachya及紫叶兔耳草L.praecox的种内最大遗传距离均小于种间最小遗传距离，具有明显的条形码间距。系统进化树显示短筒兔耳草、圆穗兔耳草、短穗兔耳草及紫叶兔耳草单独聚为一支，四川部分区域所产全缘兔耳草与革叶兔耳草聚为一支，其余全缘兔耳草聚为一支。通过ITS2二级结构发现，各基原兔耳草在茎环数目、大小及位置均有一定差异，可区分亲缘关系近的全缘兔耳草与革叶兔耳草。市场分析显示，市面上流通的兔耳草有6种基原，以全缘兔耳草使用占比最高，短筒兔耳草次之，短穗兔耳草及圆穗兔耳草较低，紫叶兔耳草及革叶兔耳草最低。结论 ITS2序列可以作为DNA条形码用来鉴别常用藏药兔耳草的基原，市场主流兔耳草品种为全缘兔耳草、短筒兔耳草及短穗兔耳草。

Identification of commonly used origins of Tibetan medicine Lagotidis Herba based on ITS2 barcode and market analysis

Objective To establish a rapid method to identify different origin of Lagotidis Herba from self-harvesting and distribution samples based on ITS2 sequences of DNA barcode technology, and analyze the mainstream varieties in the market. Method The DNA of 101 Lagotidis Herba samples was extracted by using the Plant Genomic DNA Extraction kit, and then their ITS2 sequence was amplified by PCR and sequenced in both directions to obtain the information of ITS2 sequence; Meanwhile, 28 related sequences were downloaded from GenBank. Subsequently, 129 ITS2 sequences were analyzed by MEGA6.0, and the intra-and inter-specific K2P genetic distances were calculated to construct a neighbor joining (NJ) phylogenetic tree and predict the secondary structure of ITS2. The origins of Lagotidis Herba in circulation samples were analyzed by the BLAST and NJ cluster analysis methods. Result The results showed that the maximum intraspecific genetic distance was far less than the minimum interspecific genetic distance among Lagotis brevituba, L. integra, L. ramalana, L. alutacea, L. brachystachya and L. praecox, except L. integra from some areas of Sichuan and L. alutacea, indicating there were an obvious barcoding gap among them. The NJ tree showed that L. brevituba, L. ramalana, L. brachystachya and L. praecox shared respectively a clade, and L. integra from some areas of Sichuan and L. alutacea shared a clade, and other L. integra shared a clade. ITS2 secondary structures for six kinds of Lagotidis Herba were significantly different enough to identify L. integra and L. alutacea, and the difference were the number, size and location of the stem ring. Market analysis showed that the usage rate of different origins of L. herba from high to low was L. integra, L. brevituba, L. brachystachya and L. ramalana, L. praecox and L. alutacea. Conclusion The ITS2 sequence can be used as DNA barcode to identify the origins of Lagotidis Herba accurately and quickly. Market mainstream varieties were L. integra, L. brevituba and L. brachystachya.