利用DNA条形码技术鉴定药用双子叶植物
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摘要
双子叶植物进化等级高、适应性强、种类繁多、分布广泛,具有重要的药用及生物学价值。然而传统的鉴定方法不能满足日益复杂的分类需求,食用、药用与有毒植物的鉴定存在一定困难。植物DNA条形码(DNA barcoding)技术使用短的标准分子片段作为分子标签,能够对物种进行快速、准确、有效鉴定,该技术的出现为解决这一难题提供了可能。
DNA条形码研究是目前分类学研究的热点,也是进展最迅速的学科前沿之一,该领域研究的热点和关键问题是通用条形码序列的筛选、评价和验证,但其在植物界尚未达成共识。2009年,rbcL+matK复合序列被推荐为植物通用条形码,但其物种水平鉴定成功率仅72%。2010年本课题组推荐nrDNA ITS2序列作为药用植物DNA条形码。但是这些研究中都缺乏在单一科属内大量密切相关物种的验证,DNA条形码序列搜索和验证工作还在继续。
本文分别选取了双子叶植物纲离瓣花亚纲和合瓣花亚纲的两个大科：豆科和菊科为代表进行研究,将目前业内热点候选序列matK, rbcL, psbA-trnH以及nrDNA ITS2(ITS)序列针对豆科、菊科植物分别进行比较研究,探讨在同一科属下更多近缘种存在的情况下候选序列的真实鉴定能力。
结果显示,自行设计引物的ITS2序列在广泛的实验植物范围内通用性强,种间差异较大,种间变异和的种内变异存在显著性差异,具有明显的Barcoding Gap,同时物种鉴定成功率高。在豆科植物中,ITS2能够正确鉴定24个药典种及其66个混伪品,对于196个属的1126个物种的1507份豆科植物样品,ITS2在属的鉴定水平高达100%,在物种水平也达到80%以上的高鉴定效率。ITS2序列应用于黄芪属的研究也进一步验证了该序列的高效性,ITS2能够正确鉴定黄芪属41个密切相关的药用植物物种。此外,ITS2的二级结构也具有一定的系统学及分类学意义,具有重要的应用价值。在494个属的2,315个菊科植物物种的3,490份序列中,ITS2与其它几个候选的DNA条形码序列相比,综合评价指标最优,并能够将97.4%的序列正确鉴定到属,76.4%的序列正确鉴定到种(BLAST 1方法)。
matK的鉴定能力也很强,对于409个属的1,079个豆科植物物种的1,355份序列在种和属的水平分别达到了约80%及96%,然而其通用性较差限制了该序列的广泛使用; psbA-trnH基因间隔区也表现出了较高的鉴定能力,它可以将豆科药用植物及其常见伪品共97个物种进行成功鉴别,但是该序列存在PLOY结构影响了其测序质量,而且公共数据库中该序列数据较少,未能进行大规模验证。
因此综合考虑,我们认为ITS2基因间隔区在豆科菊科植物中具有广泛“通用性”及在单一科属内的“高效性”,故推荐ITS2序列作为双子叶植物纲DNA条形码通用序列,并可考虑与其他两个序列matK, psbA-trnH组合进一步完善植物DNA条形码研究。本研究为药用植物鉴定、濒危物种保护、资源评价等提供了新的思路,并展示了DNA条形码技术实际应用的广泛前景。
With high levels of evolution, adaptation and diversities, the dicotyledon plants are widely distributed and are of great importantance of medicinal and biological values. The identification of different species is difficult especially based solely on morphological characteristics; additionally, the limitations in traditional taxonomy prevent these techniques from meeting the complicated demands of species recognition. DNA barcoding is a novel technology that uses a standard DNA sequence to facilitate simple and accurate species identification.
DNA barcoding is a rapidly developing frontier technology, it has become a hotspot and has gained worldwide attentions in the scientific academica. The studies about how to select, evaluate and verify the appropriated DNA barcode candidate has been reported increasingly.Up until now, a consensus has not been reached regarding which DNA sequences can be used as the best plant barcodes. In 2009, the DNA regions rbcL, matK have been recommended by Plant working group of CBOL (the Consortium for the Barcode of Life) as DNA barcodes for plants, yet the successful identification rate of this barcoding combination is only 73% at specific level, the work is "far from perfect". In 2010, Chen et al. compared the practicality of using the suggested barcode sequences against a large number of medicinal plants and recommended ITS2 as a DNA barcode. But the study to evaluate these regions for species identification in the large plant taxon which includes a large number of closely related species is rarely reported. Thus, works of this area keep on performing.
We tested the feasibility of using four proposed DNA regions(matK, rbcL, psbA-trnH and nrDNA ITS2, ITS) for discriminating plant species within Fabaceae (Choripetalae) and Asteraceae (Sympetalae), the two large families of dicotyledon which include a large number of closely related species.
Our results demonstrate that ITS2 was useful in regards to its universality, sequence variation, "barcoding gap" and identification capability of the Fabaceae and Asteraceae family. In Fabaceae, twenty-four species found in the Chinese Pharmacopoeia, along with another sixty-six species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1,507 sequences derived from 1,126 species belonging to 196 genera. The study of "identification of Astragalus Plants in China using ITS2" further verified the power of ITS2.41 of 47 Astragalus close related species have been identified by ITS2. Besides, ITS2 secondary structure is of significance for the systematic and taxonomic study. Therefore, ITS2 may be a useful barcode for Astragalus species, with great application value. In Asteraceae, ITS2 performed best of other markers. We also explored the species discriminating power of ITS2 on a large-scale within Asteraceae using 3,490 sequences representing 2,315 species belonging to 494 different genera and found that it correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively (BLAST 1 method).
matK is a useful barcode for Fabaceae species. Of 1,355 plant samples collected from 1,079 species belonging to 409 diverse genera, matK precisely identified approximately 80% and 96% of them at the species and genus levels, respectively. However, at present, the use of matK as a universal barcode remains problematic due to the absence of universal primers. The psbA-trnH plastid region is as a powerful marker to the authentication of Fabaceae medicinal plants. We correctly differentiate 22 species belonging to 19 genera listed in the Chinese Pharmacopoeia only using the psbA-trnH plastid region. Moreover, the psbA-trnH regions could also distinguish between 97 medicinal plant species of Fabaceae commonly used including their adulterants, and seven adulterants of outgroup species. Yet, there were some poor quality psbA-trnH sequences mainly due to the presence of long mononucleotide repeats. In addition, the psbA-trnH sequences in public database is not much, large-scale verification can not be done.
Altogether, ITS2 region has already been recommended as a core marker for taxonomic classification and barcoding in dicotyledon based on its universal and high species identification rate within Fabaceae and Asteraceae. Furthermore, we propose that matK and psbA-trnH be used in combination with ITS2 as standard barcodes. This approach will significantly broaden the application of DNA barcoding to resolve classification problems at the genera and species levels of dicotyledon and provide public with a powerful tool for practical use.
DNA条形码研究是目前分类学研究的热点,也是进展最迅速的学科前沿之一,该领域研究的热点和关键问题是通用条形码序列的筛选、评价和验证,但其在植物界尚未达成共识。2009年,rbcL+matK复合序列被推荐为植物通用条形码,但其物种水平鉴定成功率仅72%。2010年本课题组推荐nrDNA ITS2序列作为药用植物DNA条形码。但是这些研究中都缺乏在单一科属内大量密切相关物种的验证,DNA条形码序列搜索和验证工作还在继续。
本文分别选取了双子叶植物纲离瓣花亚纲和合瓣花亚纲的两个大科：豆科和菊科为代表进行研究,将目前业内热点候选序列matK, rbcL, psbA-trnH以及nrDNA ITS2(ITS)序列针对豆科、菊科植物分别进行比较研究,探讨在同一科属下更多近缘种存在的情况下候选序列的真实鉴定能力。
结果显示,自行设计引物的ITS2序列在广泛的实验植物范围内通用性强,种间差异较大,种间变异和的种内变异存在显著性差异,具有明显的Barcoding Gap,同时物种鉴定成功率高。在豆科植物中,ITS2能够正确鉴定24个药典种及其66个混伪品,对于196个属的1126个物种的1507份豆科植物样品,ITS2在属的鉴定水平高达100%,在物种水平也达到80%以上的高鉴定效率。ITS2序列应用于黄芪属的研究也进一步验证了该序列的高效性,ITS2能够正确鉴定黄芪属41个密切相关的药用植物物种。此外,ITS2的二级结构也具有一定的系统学及分类学意义,具有重要的应用价值。在494个属的2,315个菊科植物物种的3,490份序列中,ITS2与其它几个候选的DNA条形码序列相比,综合评价指标最优,并能够将97.4%的序列正确鉴定到属,76.4%的序列正确鉴定到种(BLAST 1方法)。
matK的鉴定能力也很强,对于409个属的1,079个豆科植物物种的1,355份序列在种和属的水平分别达到了约80%及96%,然而其通用性较差限制了该序列的广泛使用; psbA-trnH基因间隔区也表现出了较高的鉴定能力,它可以将豆科药用植物及其常见伪品共97个物种进行成功鉴别,但是该序列存在PLOY结构影响了其测序质量,而且公共数据库中该序列数据较少,未能进行大规模验证。
因此综合考虑,我们认为ITS2基因间隔区在豆科菊科植物中具有广泛“通用性”及在单一科属内的“高效性”,故推荐ITS2序列作为双子叶植物纲DNA条形码通用序列,并可考虑与其他两个序列matK, psbA-trnH组合进一步完善植物DNA条形码研究。本研究为药用植物鉴定、濒危物种保护、资源评价等提供了新的思路,并展示了DNA条形码技术实际应用的广泛前景。
With high levels of evolution, adaptation and diversities, the dicotyledon plants are widely distributed and are of great importantance of medicinal and biological values. The identification of different species is difficult especially based solely on morphological characteristics; additionally, the limitations in traditional taxonomy prevent these techniques from meeting the complicated demands of species recognition. DNA barcoding is a novel technology that uses a standard DNA sequence to facilitate simple and accurate species identification.
DNA barcoding is a rapidly developing frontier technology, it has become a hotspot and has gained worldwide attentions in the scientific academica. The studies about how to select, evaluate and verify the appropriated DNA barcode candidate has been reported increasingly.Up until now, a consensus has not been reached regarding which DNA sequences can be used as the best plant barcodes. In 2009, the DNA regions rbcL, matK have been recommended by Plant working group of CBOL (the Consortium for the Barcode of Life) as DNA barcodes for plants, yet the successful identification rate of this barcoding combination is only 73% at specific level, the work is "far from perfect". In 2010, Chen et al. compared the practicality of using the suggested barcode sequences against a large number of medicinal plants and recommended ITS2 as a DNA barcode. But the study to evaluate these regions for species identification in the large plant taxon which includes a large number of closely related species is rarely reported. Thus, works of this area keep on performing.
We tested the feasibility of using four proposed DNA regions(matK, rbcL, psbA-trnH and nrDNA ITS2, ITS) for discriminating plant species within Fabaceae (Choripetalae) and Asteraceae (Sympetalae), the two large families of dicotyledon which include a large number of closely related species.
Our results demonstrate that ITS2 was useful in regards to its universality, sequence variation, "barcoding gap" and identification capability of the Fabaceae and Asteraceae family. In Fabaceae, twenty-four species found in the Chinese Pharmacopoeia, along with another sixty-six species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1,507 sequences derived from 1,126 species belonging to 196 genera. The study of "identification of Astragalus Plants in China using ITS2" further verified the power of ITS2.41 of 47 Astragalus close related species have been identified by ITS2. Besides, ITS2 secondary structure is of significance for the systematic and taxonomic study. Therefore, ITS2 may be a useful barcode for Astragalus species, with great application value. In Asteraceae, ITS2 performed best of other markers. We also explored the species discriminating power of ITS2 on a large-scale within Asteraceae using 3,490 sequences representing 2,315 species belonging to 494 different genera and found that it correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively (BLAST 1 method).
matK is a useful barcode for Fabaceae species. Of 1,355 plant samples collected from 1,079 species belonging to 409 diverse genera, matK precisely identified approximately 80% and 96% of them at the species and genus levels, respectively. However, at present, the use of matK as a universal barcode remains problematic due to the absence of universal primers. The psbA-trnH plastid region is as a powerful marker to the authentication of Fabaceae medicinal plants. We correctly differentiate 22 species belonging to 19 genera listed in the Chinese Pharmacopoeia only using the psbA-trnH plastid region. Moreover, the psbA-trnH regions could also distinguish between 97 medicinal plant species of Fabaceae commonly used including their adulterants, and seven adulterants of outgroup species. Yet, there were some poor quality psbA-trnH sequences mainly due to the presence of long mononucleotide repeats. In addition, the psbA-trnH sequences in public database is not much, large-scale verification can not be done.
Altogether, ITS2 region has already been recommended as a core marker for taxonomic classification and barcoding in dicotyledon based on its universal and high species identification rate within Fabaceae and Asteraceae. Furthermore, we propose that matK and psbA-trnH be used in combination with ITS2 as standard barcodes. This approach will significantly broaden the application of DNA barcoding to resolve classification problems at the genera and species levels of dicotyledon and provide public with a powerful tool for practical use.
引文
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