杜鹃花属药用植物DNA条形码的鉴定研究
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摘要
DNA条形码技术是利用一段(或几段)通用的、相对较短且具有足够变异的易扩增的DNA片段对物种进行鉴定,它是依据DNA片段在物种内的特异性和种间的多样性而创建的一种新的物种鉴别方法,能够实现对物种的快速和较客观的鉴定。本文选用生物多样性丰富且药用资源较多的杜鹃花属植物作为对象进行DNA条形码鉴定研究。
为评价和比较几个热点推荐的DNA条形码候选序列对杜鹃花属药用植物的鉴定作用,本研究首先对杜鹃花属38个物种68份样品的psbA-trnH、rbcL.matK和核ITS2序列进行PCR扩增和测序,比较各序列扩增成功率和测序成功率、种内和种间变异,进行barcoding gap分析,采用BLAST1和Nearest Distance方法评价四个不同序列的鉴定能力。结果显示：psbA-trnH序列在所研究的杜鹃花属药用植物中的测序成功率为86.8%,rbcL序列为89.7%,核ITS2序列仅为50%；psbA-trnH序列提供了最大种间变异且Wilcoxon检验表明其种间最小变异明显大于其种内最大变异,较有利于物种的鉴别；barcoding gap与其他DNA条形码候选序列相比具有明显的优势,psbA-trnH序列在杜鹃花属(59个样品)中的鉴定成功率可达到100%,远远高于rbcL(59%)和核ITS2(41.2%)的鉴定成功率。将psbA-trnH序列扩大应用至94个样品,依然具有93.6%的鉴定成功率,即使对于其中不能鉴定到种的6个样本,psbA-trnH序列亦可以缩小其分类范围至一个亚属甚至是一个组或一个亚组,将其确定到一个较小的分类范围,具有一定鉴定意义。因此,psbA-trnH字列可以作为这四个序列中杜鹃花属植物条形码首先推荐的序列。
有关杜鹃花属的属下分类一直存在争议,由于psbA-trnH序列在杜鹃花属物种的鉴定方面表现出了较好的鉴定能力,本研究运用psbA-trnH1序列的测序结果,选用杜鹃花属7个亚属37个物种的样本,用MEGA4.0进行相关数据分析,并构建聚类树,对杜鹃花属各个亚属的分类地位进行了探讨,为进一步深入研究杜鹃花属植物的亲缘关系提供分子系统学证据。结果表明杜鹃花属37个种的样本聚为7枝,与《中国植物志》上分类系统相符,支持《中国植物志》杜鹃花属下亚属的分类系统。这也从系统进化的角度,证实了psbA-trnH序列.在杜鹃花属植物分类和鉴定中的作用。
杜鹃花属药用植物同名异物、同物异名现象严重,混淆品和伪品较多,ITS2片段在中药材的物种鉴定方面具有潜在的研究价值。本文考察利用ITS2序列对杜鹃花属10种常用中药原植物进行鉴别的可行性,为ITS2序列准确鉴别这些药用植物提供分子证据,结果表明ITS2作为DNA条形码序列能够准确区别10种杜鹃花属药用植物。
DNA barcoding is a new biological tool for accurately and objectively identifying species by using short and standard DNA regions that can be amplified easily by PCR. One of the problems in plant DNA barcode reseaches was that the previous studies were mainly carried out on a large scale with only a few species within one genus and rarely on some specific genus with much sister species, which result in a superficially high rate of discrimination and low credibility of the reference database. Rhododendron is a group of famous landscape plants with high medicinal and ornamental value. However, there is no simple and universal way to discriminate various species of this group. DNA barcoding technique is a new biological tool that can accurately and objectively identify species by using short and standard DNA regions. The CBOL PWG established the use of matK+ rbcL as core barcodes and ITS2 as one of the supplementary loci for differentiating plants at the Third International Barcoding Conference. To choose a suitable DNA marker to authenticate Rhododendron species, four candidate DNA barcodes (rbcL, matK, psbA-trnH, ITS2 intergenic spacer) were tested on sixty-eight samples of thirty-eight species. We found that the psbA-trnH candidate barcode yielded 86.8% sequencing efficiency. The highest interspecific divergence was provided by the psbA-trnH intergenic spacer based on six parameters and Wilcoxon signed rank tests. Although there was not a clear barcoding gap, Wilcoxon two sample tests indicated that the interspecific divergence of the psbA-trnH intergenic spacer was significantly higher than the relevant intraspecific variation. The psbA-trnH DNA barcode possessed the highest species identification efficiency at 100% by BLAST1 method, followed by rbcL only 59%. The present results showed that psbA-trnH intergenic spacer was the most promising one of the four markers for barcoding Rhododendron species. To further evaluate the ability of the psbA-trnH marker to discriminate closely related species, the samples were expanded to ninety-four samples of fifty-three species in the genus and the rate of successful identification was 93.6%. Among the unidentifiable individuals of three pair samples, every pair comes from a same subgenus or section with complicated background in taxa and this shows that the psbA-trnH sequence could narrow the sample to a small taxa area and reveal the existing of complicated group in taxa.In conclusion, the psbA-trnH intergenic region is a valuable DNA marker for identifying Rhododendron species.
Since the genus was established by Linnaeus, various methods have been constantly used to revise its classification system on the basis of morphology, cytology, chemotaxonomy and molecular taxonomy. However, the Rhododendron genus still has some problems at various systematic classification levels; there is no simple and universal way to discriminate various species within the genus. Phylogenetic relationships within the genus Rhododendron were studied using psbA-trnff intergenic spacer seqences. In the analysis, thirty-five Rhododendron species, which belong to seven subgenus according to taxonomy system of Sleumer in "Flora of China" were analyzed. The results indicated that:all species are grouped into seven clades and all samples come from every same subgenus formed into a monophyletic clade with higher bootstrap value which supported the taxonomy system of Sleumer in "Flora of China". Pseudorhodorastrum as an independent subgenus was supported. The opinion of Chamberlian proposed the subg. Pseudazalea should be placed in subg. Rhododendron as a subsection Trichoclada was not supported by the reconstructed phylogenetic tree.
The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. To discriminate ten medicinal plants in Rhododendron us ing ITS2 sequence, twenty-three sequences were analyzed by means of CLUSTRAL X and MEGA softwares and the phylogenetic tree was constructed using UPGMA and in the cluster dendrogram ten species of Rhododendron showed monophyletic. The result showed that ITS2 sequence can be used to correctly identify ten medicinal plants in Rhododendron, and it has certain application value in the identification of traditional Chinese medicine in Rhododendron.
为评价和比较几个热点推荐的DNA条形码候选序列对杜鹃花属药用植物的鉴定作用,本研究首先对杜鹃花属38个物种68份样品的psbA-trnH、rbcL.matK和核ITS2序列进行PCR扩增和测序,比较各序列扩增成功率和测序成功率、种内和种间变异,进行barcoding gap分析,采用BLAST1和Nearest Distance方法评价四个不同序列的鉴定能力。结果显示：psbA-trnH序列在所研究的杜鹃花属药用植物中的测序成功率为86.8%,rbcL序列为89.7%,核ITS2序列仅为50%；psbA-trnH序列提供了最大种间变异且Wilcoxon检验表明其种间最小变异明显大于其种内最大变异,较有利于物种的鉴别；barcoding gap与其他DNA条形码候选序列相比具有明显的优势,psbA-trnH序列在杜鹃花属(59个样品)中的鉴定成功率可达到100%,远远高于rbcL(59%)和核ITS2(41.2%)的鉴定成功率。将psbA-trnH序列扩大应用至94个样品,依然具有93.6%的鉴定成功率,即使对于其中不能鉴定到种的6个样本,psbA-trnH序列亦可以缩小其分类范围至一个亚属甚至是一个组或一个亚组,将其确定到一个较小的分类范围,具有一定鉴定意义。因此,psbA-trnH字列可以作为这四个序列中杜鹃花属植物条形码首先推荐的序列。
有关杜鹃花属的属下分类一直存在争议,由于psbA-trnH序列在杜鹃花属物种的鉴定方面表现出了较好的鉴定能力,本研究运用psbA-trnH1序列的测序结果,选用杜鹃花属7个亚属37个物种的样本,用MEGA4.0进行相关数据分析,并构建聚类树,对杜鹃花属各个亚属的分类地位进行了探讨,为进一步深入研究杜鹃花属植物的亲缘关系提供分子系统学证据。结果表明杜鹃花属37个种的样本聚为7枝,与《中国植物志》上分类系统相符,支持《中国植物志》杜鹃花属下亚属的分类系统。这也从系统进化的角度,证实了psbA-trnH序列.在杜鹃花属植物分类和鉴定中的作用。
杜鹃花属药用植物同名异物、同物异名现象严重,混淆品和伪品较多,ITS2片段在中药材的物种鉴定方面具有潜在的研究价值。本文考察利用ITS2序列对杜鹃花属10种常用中药原植物进行鉴别的可行性,为ITS2序列准确鉴别这些药用植物提供分子证据,结果表明ITS2作为DNA条形码序列能够准确区别10种杜鹃花属药用植物。
DNA barcoding is a new biological tool for accurately and objectively identifying species by using short and standard DNA regions that can be amplified easily by PCR. One of the problems in plant DNA barcode reseaches was that the previous studies were mainly carried out on a large scale with only a few species within one genus and rarely on some specific genus with much sister species, which result in a superficially high rate of discrimination and low credibility of the reference database. Rhododendron is a group of famous landscape plants with high medicinal and ornamental value. However, there is no simple and universal way to discriminate various species of this group. DNA barcoding technique is a new biological tool that can accurately and objectively identify species by using short and standard DNA regions. The CBOL PWG established the use of matK+ rbcL as core barcodes and ITS2 as one of the supplementary loci for differentiating plants at the Third International Barcoding Conference. To choose a suitable DNA marker to authenticate Rhododendron species, four candidate DNA barcodes (rbcL, matK, psbA-trnH, ITS2 intergenic spacer) were tested on sixty-eight samples of thirty-eight species. We found that the psbA-trnH candidate barcode yielded 86.8% sequencing efficiency. The highest interspecific divergence was provided by the psbA-trnH intergenic spacer based on six parameters and Wilcoxon signed rank tests. Although there was not a clear barcoding gap, Wilcoxon two sample tests indicated that the interspecific divergence of the psbA-trnH intergenic spacer was significantly higher than the relevant intraspecific variation. The psbA-trnH DNA barcode possessed the highest species identification efficiency at 100% by BLAST1 method, followed by rbcL only 59%. The present results showed that psbA-trnH intergenic spacer was the most promising one of the four markers for barcoding Rhododendron species. To further evaluate the ability of the psbA-trnH marker to discriminate closely related species, the samples were expanded to ninety-four samples of fifty-three species in the genus and the rate of successful identification was 93.6%. Among the unidentifiable individuals of three pair samples, every pair comes from a same subgenus or section with complicated background in taxa and this shows that the psbA-trnH sequence could narrow the sample to a small taxa area and reveal the existing of complicated group in taxa.In conclusion, the psbA-trnH intergenic region is a valuable DNA marker for identifying Rhododendron species.
Since the genus was established by Linnaeus, various methods have been constantly used to revise its classification system on the basis of morphology, cytology, chemotaxonomy and molecular taxonomy. However, the Rhododendron genus still has some problems at various systematic classification levels; there is no simple and universal way to discriminate various species within the genus. Phylogenetic relationships within the genus Rhododendron were studied using psbA-trnff intergenic spacer seqences. In the analysis, thirty-five Rhododendron species, which belong to seven subgenus according to taxonomy system of Sleumer in "Flora of China" were analyzed. The results indicated that:all species are grouped into seven clades and all samples come from every same subgenus formed into a monophyletic clade with higher bootstrap value which supported the taxonomy system of Sleumer in "Flora of China". Pseudorhodorastrum as an independent subgenus was supported. The opinion of Chamberlian proposed the subg. Pseudazalea should be placed in subg. Rhododendron as a subsection Trichoclada was not supported by the reconstructed phylogenetic tree.
The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. To discriminate ten medicinal plants in Rhododendron us ing ITS2 sequence, twenty-three sequences were analyzed by means of CLUSTRAL X and MEGA softwares and the phylogenetic tree was constructed using UPGMA and in the cluster dendrogram ten species of Rhododendron showed monophyletic. The result showed that ITS2 sequence can be used to correctly identify ten medicinal plants in Rhododendron, and it has certain application value in the identification of traditional Chinese medicine in Rhododendron.
引文
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