基于DNA条形码对凉山虫草及近缘物种和其混伪品的分子鉴定

目的：结合ITS基因和COI基因对《四川省中药材标准》收录的凉山虫草和近缘物种及当地市场上出现的混伪品进行分子鉴定，考察其方法的可行性。方法：对收集的28份样品通过DNA提取、PCR扩增，分别获得其ITS 序列和COI 序列。用Codon Code Aligner V3.7.1，Mega 5.0等软件进行比对分析，构建基于ITS 序列和COI 序列的样品的NJ 树，进行聚类分析。结果：凉山虫草和近缘物种及混伪品寄生真菌的ITS 序列种间最小K-2P距离大于各物种种内最大K-2P距离：种内变异小，种间差异较大，从而基于ITS序列的NJ 树能将凉山虫草与其他近缘物种及其混伪品很好地归属；凉山虫草和近缘物种及其混伪品寄主昆虫的COI序列种间最小K-2P距离大于各物种种内最大K-2P距离：种内变异小，种间差异较大，通过遗传距离及NJ树的分析能将凉山虫草与其他近缘物种其及其混伪品及其他近缘物种很好地区分。结论：结合ITS序列和COI序列的DNA条形码技术可以很好地鉴定凉山虫草与其近缘物种及其混伪品，并对虫草属的系统学和分类研究提供了技术支持。

Molecular Identification of Metacordyceps Liangshanensis, Its Adulterants and Its Relative Species Based on DNA Barcode

This study was aimed to identify and distinguish Metacordyceps liangshanensis recorded by the Sichuan Province Traditional Chinese Medicine Standard from its adulterants and its relative species by combining ITS and COI barcode sequences in order to study the feasibility of this new method. After extracting DNA of 28 species of Cordyceps samples, DNA were amplified and sequenced. And then, ITS and COI sequences were received. CodonCode Aligner V3.7.1 and Mega 5.0 were used to analyze the variable site and construct the N-J tree. The results showed that the minimum ITS inter-specific K-2P distance was relatively higher than the maximum intra-specific K-2P distance. The inter-specific sequence divergence between M. liangshanensis and its adulterants exhibited high while intra-specific sequence divergence exhibited low. And COI one was the same case. N-J tree of both ITS and COI indicated that same genus belonged together and each species belonged to relatively independent branch. It was concluded that based on the ITS and COI gene, the technology of DNA barcode can be an excellent identification of M. liangshanensis, its adulterants and its relative species. It provided technical support for the further research on species molecular identification and phylogenetics of Cordyceps.