莪术基原植物DNA条形码序列的筛选与鉴定

Screening and identification on DNA barcoding sequences of original plants of Curcumae Rhizoma

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1)ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642~645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。

In order to find a rapid and efficient identification method for three original species of Curcumae Rhizoma,identification efficiency of different DNA barcoding sequences were evaluated in present study.Totally nine samples of three different species of Curcuma were collected.After DNA extraction,PCR amplification and sequencing,seven kinds of DNA barcoding sequences,including ITS,ITS2,matK,psbA-trnH,trnL-trnF,rpoB and atpB-rbcL,were firstly compared in terms of success rate of PCR amplification and characteristics of sequences.Then,by means of variation site analysis and genetic distance calculation,these seven kinds of DNA barcoding sequences were further evaluated.Finally,the unidentified samples were identified by the phylogenetic tree based on the chosen DNA barcoding sequences.The results were as follows:(1)ITS,ITS2 and matK barcoding sequences were inapplicable due to the low success rate of PCR amplification and sequencing;The variation information of psbA-trnH,trnL-trnF and rpoB was insufficient to distinguish three different original species of Curcumae Rhizoma;Only atpB-rbcL barcoding sequence was 642-645 bp in length with 29.0%-29.9%GC content and 11 variation sites,for which,the three species of Curcumae Rhizoma could be distinguished only by atpB-rbcL barcoding sequence.(2)According to the phylogenetic tree based on atpB-rbcL sequence,the unidentified samples were identified as Curcuma wenyujin.In conclusion,atpB-rbcL sequence could be used as a standard sequence for the rapid and efficient identification of original species of Curcumae Rhizoma.