基于DNA条形码技术的重楼栽培品基原鉴定

目的：基于DNA条形码技术鉴定重楼栽培品的基原，并比较同物种重楼栽培品与野生品的DNA条形码差异。方法：通过聚合酶链式反应（PCR）对重楼的ITS和ITS2序列进行扩增；通过与GenBank数据库进行Blast比对，确定收集重楼样品的物种；利用距离法和建树法比较重楼样品种内和种间的亲缘关系；比较不同物种样品之间，以及同物种样品的野生品与栽培品之间的碱基差异。结果：收集重楼样品经Blast比对共有10个物种，分别为宽瓣重楼、毛重楼、七叶一枝花、花叶重楼、独龙重楼、五指莲重楼、西藏延龄草、平伐重楼、华重楼、南重楼；除南重楼的ITS序列与毛重楼的平均种间遗传距离小于南重楼的最大种内遗传距离外，各个物种的ITS和ITS2序列的最大种内遗传距离均小于其最小种间平均遗传距离，通过建立NJ树聚类分析，重楼样品的10个物种中相同物种聚类，且具有良好的单系性；从碱基比较结果上看，野生重楼样品与栽培重楼样品的碱基差异很小，甚至完全一致。结论：市场流通重楼药材的性状多样且差异很大并非由于人工驯化造成，而是由于重楼栽培基原的混乱造成的，且多数流通样品为非《中华人民共和国药典》收载物种，通过DNA条形码技术为重楼属药用植物鉴别开辟了一条新途径。

Objective:To identify the origin of Paridis Rhizoma samples based on DNA barcoding technology,and to compare differences between the wild and cultivated samples in DNA barcoding.Methods:ITS sequences and ITS2 sequences of Paridis Rhizoma samples were amplified with polymerase chain reaction (PCR),which were blasted with GenBank database to determine species.The intraspecific and interspecific genetic relationship was evaluated by the methods of nearest distance and NJ tree by comparing the base differences among diverse species and among wild and cultivated samples.Results:There were 10 species identified in 60 samples of Paridis Rhizoma,and they were Paris polyphylla Smith var.yunnanensis,Paris polyphylla Smith var.chinensis,Paris polyphylla,Paris vietnamensis,Paris mairei,Paris marmorata,Paris dulongensis,Paris axialis,Trillium govanianum and Paris vaniotii.For each of the different species'ITS and ITS2 sequences,the largest genetic distance of intraspecific was less-than the minimum average genetic distances,except the ITS sequence of Paris vietnamensis.The analysis of NJ trees indicated that the same species clustered together and revealed better monophyly within 10 species.Difference between the wild and cultivated samples was tiny and the samples were even the same.Conclusion:It was not the cultivation but the origin confusion which led to the disorder of the commercial market of Paridis Rhizoma.And majority of samples in the market are not species adopted in Pharmacopoeia of the People's Republic of China.DNA barcoding technology offered a new approach for identification of Paris plants.