重楼属药用植物DNA条形码鉴定研究

DNA barcoding the medicinal plants of the genus Paris

为评价DNA条形码候选序列对重楼属药用植物的鉴定作用,探讨重楼属药用植物鉴定新方法,本研究对重楼属11个物种17份样品的psbA-trnH、rpoB、rpoC1、rbcL、matK和核ITS2序列进行PCR扩增和测序,比较各序列扩增和测序效率、种内和种间变异,进行barcoding gap分析,采用BLAST1和Nearest Distance方法评价不同序列的鉴定能力。结果显示,ITS2序列在所研究的重楼属药用植物中的扩增和测序效率均为100%,其种内种间变异、barcoding gap与其他DNA条形码候选序列相比具有明显的优势,ITS2序列在重楼属中的鉴定成功率达到100%,而生物条形码协会(CBOL)植物工作组推荐的matK和rbcL序列的鉴定成功率分别为52.9%和5.9%,二者联合鉴定能力没有提高,对于ITS2序列扩大至29个物种67份样品依然具有100%的鉴定成功率。实验结果表明,ITS2序列能够准确鉴定重楼属药用植物,可以作为潜在的药用植物通用条形码序列。

DNA barcoding is a technique in which species identification and discovery are performed by using short and standard fragments of DNA sequences. In this study, eleven species of Paris, including seven varieties, were sampled. Five chloroplast sequences, psbA-trnH, rpoB, rpoC1, rbcL, matK, and one nuclear marker, the second internal transcribed spacer （ITS2） of ribosomal DNA, were amplified and sequenced. The PCR amplification and sequencing efficiency, intra-and inter-specific divergence and barcoding gap were used to evaluate different loci, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The ITS2 sequences in the studied samples of Paris were amplified and sequenced successfully using primers designed by our group, while matK showed low level in the amplification and psbA-trnH was difficult for sequencing because of over 800 bp and poly （A） structure. Analysis of the intra-and inter-specific divergence and barcoding gap showed ITS2 was superior to other loci. The ITS2 showed a much higher percentage of success （100%） in identification than other five loci, none of which indicated more than 50% except matK （52.9%）. The 2-locus combination of rbcL＋matK didn’t improve ability of authentication. In addition, the rate of successful identification with ITS2 kept 100% when the samples were expanded to 67 samples of 29 species. In conclusion, ITS2 can be used to correctly identify medicinal plants of Paris, and it will be a potential DNA barcode for identifying medicinal plants of other taxa.