基于通用DNA条形码序列的黄精属药用植物分子鉴定

目的 分析4个通用植物DNA条形码序列(trnH-psbA、matK、rbcL和ITS2)及其组合对黄精属药用植物的物种鉴定分辨率,挖掘适用于黄精属种间鉴定的高分辨率分子标记。方法 以《中国药典》2020年版中收录的黄精属药用植物黄精Polygonatum sibiricum、滇黄精P. kingianum、多花黄精P. cyrtonema、玉竹P. odorati及其地方常见同属替代品、混伪品共12种79个野生个体为对象,将4个通用DNA条形码序列独立、联合分析,评估其种间、种内变异情况,并分别基于建树法(tree-based method)和PWG距离法(PWG-distance method)评估不同条形码及其组合的物种鉴定分辨率。结果 ITS2序列扩增成功率低,trnH-psbA、matK、rbcL序列的引物在黄精属植物中通用性较好;3组叶绿体序列的种间变异依次为matK＞trnH-psbA＞rbcL,种内变异差异不显著,种间、种内遗传距离无明显的Barcoding gap;各条形码独立及联合分析的物种鉴定分辨率普遍偏低,其中,组合条形码trnH-psbA＋matK＋rbcL在建树法分析中的分辨率最高,为25%,trnH-psbA＋rbcL在距离法分析中的分辨率最高,为50%。结论 4个通用DNA条形码序列及其组合都并非黄精属药用植物不同种间有效区分鉴定的理想分子标记,但多序列联合分析能在一定程度上提高物种鉴定成功率。

Molecular authentication of medicinal Polygonatum Species utilizing the universal DNA barcode sequences

Objective To investigate the species discrimination power of the four universal plant DNA barcodes (trnH-psbA, matK, rbcL and ITS2) and corresponding multi-barcode combinations in Polygonatum, and to explore high-resolution molecular markers suitable for Polygonatum. Methods Seventy-nine wild individuals from 12 species, representing all the four medicinal species of Polygonatum (P. sibiricum, P. kingianum, P. cyrtonema, P. odoratum) included in the Chinese Pharmacopoeia (2020 Edition) and their local commonly used substitutions and inauthentic adulterants, were sampled. The interspecific and intraspecific genetic variation were estimated, tree-based and PWG-distance methods were applied to evaluate the species discrimination efficiency of each barcode sequence and their combinations. Results The primers of trnH-psbA, matK and rbcL all showed good universality while most of the individuals failed to obtain ITS2 sequence in PCR amplification. The interspecific genetic variation of the three chloroplast sequences was matK>trnH-psbA>rbcL, while their intraspecific genetic difference was not significant, and no obvious Barcoding gap was detected. All these barcode sequences including their combinations only get limited species resolution. Among which, the combination of trnH-psbA+matK+rbcL possessed the best species-resolving power of 25% in tree-based method, trnH-psbA+rbcL showed the highest resolution degree of 50% in PWG-distance method. Conclusion None of the four barcode sequences nor their combinations were ideal molecular markers to address the problems of medicinal Polygonatum species authentication. Nonetheless, as the number of sequence increases, the degree of species resolution improves.