基于ITS 2序列的15种淫羊藿属药用植物分类鉴定研究

Taxonomic Identification Analysis of 15 Medicinal Plants of Epimedium based on ITS 2 Sequence

为探讨内转录间隔区2(ITS2)序列及其二级结构在15种淫羊藿属(Epimedium)中的鉴定效率。利用试剂盒提取叶片总DNA,对ITS2序列进行PCR扩展与测序,计算K2P遗传距离,构建系统发育树,预测其二级结构。结果表明,ITS2序列在15种淫羊藿属植物中的通用性好,扩增成功率与测序成功率均为100%。从K2P种间和种内遗传距离看,ITS2在淫羊藿属中的种间变异较小,部分种类种内遗传距离大于种间遗传距离,鉴定成功率约为60%。采用比对法(Blast)分析,ITS2鉴定效率为75%。由ITS2系统进化树可知,淫羊藿属与鬼臼属各为一支,易于分开。除薄叶淫羊藿、竹山淫羊藿、巫山淫羊藿外,其余种类均能较好的进行聚类;除粗毛淫羊藿、保靖淫羊藿外其余均不能与NCBI下载到的序列进行较好聚类。研究表明,15种淫羊藿属植物ITS2序列在ML系统进化树中具较好的鉴别能力,但淫羊藿属各样本种内与种间遗传较为混乱,遗传距离复杂,ITS2二级结构形态结构亦较为相似,将各种间进行区分存在一定难度,不适合单独作为该属条形码使用。

In order to explore the efficiency of identification for the appraisal of Epimedium genera of medicinal plants in the internal transcribed spacer(ITS 2)2 sequence and secondary structure in 15 Epimedium species.The Extraction Kit was used to extract the total DNA,PCR expansion and sequencing of ITS 2 s,calculate K 2 P distance,building phylogenetic trees,predict its secondary structure.The results showed that ITS 2 had good generality in 15 species of Epimedium,the success rate of amplification and sequencing were 100%.From K 2 P interspecific and intraspecific genetic distance show that,the interspecific variation of ITS 2 in Epimedium is small,some species in genetic distance was greater than the interspecific genetic distance,appraisal success rate was about 60%.By using comparison method(Blast)analysis,ITS 2 appraisal efficiency was 75%.According to the evolution tree of ITS 2 system,Epimedium genera and ghost mortar respectively,easy to divide.All the other species could cluster well except the E.membranaceum,E.zhushanense and E.wushanense.Except for E.acuminatum and E.baojingense,all the others could not cluster well with the sequences downloaded from NCBI.The research showed that the ITS 2 s of 15 species of Epimedium had good identification ability in the ML phylogenetic tree,but the intra-and inter-species inheritance of each sample of Epimedium was relatively chaotic,the genetic distance was complex,and the morphology structure of ITS 2 secondary structure was also relatively similar.Therefore,it was difficult to distinguish all kinds of ITS 2,which was not suitable for the DNAbarcoding of Epimedium alone.