中药辛夷及其近缘种的DNA条形码分子鉴定

Molecular Identification of Magnoliae Flos and Its Relatives Based on DNA Barcoding

    摘要
  • 摘要:
      目的  应用DNA条形码技术对中药辛夷及其近缘种进行分子鉴定, 建立快速、准确、便捷的辛夷药材鉴定方法。
      方法  提取21份辛夷药材基原植物及其近缘种基因组DNA, 扩增ITS、ITS2、psbA-trnH、matK和rbcL基因序列并进行双向测序; 运用MEGA7.0软件进行序列比对, 基于Kimura-2-Parameter(K2P)模型计算种内、种间的遗传距离以评估Barcoding gap, 构建邻接(NJ)系统发育树反应鉴定结果。
      结果  5个引物序列中, psbA-trnH和matK序列的2个引物能正常扩增目的基因, 测序成功率均大于98%, rbcL测序成功率较低, 只有38.1%;其中matK具有最多的变异位点, psbA-trnH较matK和rbcL的Barcoding gap明显; NJ系统发育树显示, psbA-trnH+matK组合条形码序列能够准确鉴别9个品种, 将辛夷药材及其近缘种很好地区分, 并首次将其应用到玉兰嫁接砧木品种的分子鉴定。
      结论  应用psbA-trnH+matK的序列组合能够实现辛夷药材的准确鉴定, 并且可以应用于玉兰嫁接砧木品种的快速鉴别。

     

    Abstract:
      OBJECTIVE  To identify Magnoliae Flos and its related species using DNA barcoding technology, so as to establish a rapid, accurate and convenient method for identification of Magnoliae Flos.
      METHODS  Genomic DNA of 21 Magnoliae Flos and their related species were extracted, and ITS, ITS2, psbA-trnH, matK and rbcL gene sequences were amplified and sequenced. MEGA7.0 software was used for sequence comparison, and the intraspecific and interspecific genetic distance was calculated based on Kimura-2-Parameter (K2P) model to evaluate Barcoding gap, and the NJ phylogenetic trees were constructed to react identification results.
      RESULTS  Among the 5 primers, psbA-trnH and matK could normally amplify the target gene, and the success rate of them was higher than 98%, while the success rate of rbcL sequencing was 38.1% only. The matK sequence had the highest number of variation sites, and psbA-trnH had the most prominent species discriminatory on Barcoding gap analysis relative to matK and rbcL. The NJ phylogenetic tree showed that psbA-trnH+matK barcode sequence could accurately identify 9 varieties, and distinguish Magnoliae Flos and its relatives. In addition, we applied it to the molecular identification of Magnolia grafting rootstocks for the first time.
      CONCLUSION  The sequence combination of psbA-trnH+matK can realize the accurate identification of Magnoliae Flos, and can be applied to the rapid identification of Magnolia grafting rootstocks.

     

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