基于芸香科、天南星科的植物DNA条形码通用序列研究
摘要
目的：对比目前热点植物DNA条形码候选序列,试图筛选出植物界的通用条形码序列。
方法：选取天南星科98属223种490个样本和芸香科72属192种300个样本,针对目前热点条形码序列进行验证,考察的指标为样本的PCR扩增效率；候选序列种间、种内变异情况的6个“阈值”、种间和种内变异的"barcoding gap"以及"BLAST 1法”和“最近距离法”测得的种、属水平的鉴定成功率。并将rbcL、matK序列扩大到整个维管植物超过27000个样本,在更广泛的类群更多近缘物种存在时判断各自的鉴定能力。
结果：在天南星样本的各项考察指标中matK序列表现最为优异,进而扩大到维管植物中考察发现,matK序列在全体15765样本的鉴定效率为70.1%,而在考察的1674个属(属内物种数≥2)中,72.7%的属鉴定效率≥70%,12.7%的属鉴定效率≤40%;rbcL序列在全体11257样本的鉴定效率为61.6%,而在考察的1619个属(属内物种数≥2)中,56.4%的属鉴定效率≥70%,21.2%的属鉴定效率≤40%。由于matK、rbcL序列存在缺陷,我们在芸香科中对比陈士林等人的推荐ITS2和目前的热点条形码序列,发现ITS2序列在芸香科大量近缘属种存在时各考察指标则优于其他序列,同时基于课题组姚辉等人关于ITS2在大样本量的研究结果发现ITS2序列能较好的鉴定matK+rbcL复合序列无法鉴定的属。
结论：相对于CO1基因在动物条形码研究中的优异表现,植物条形码研究中目前所有的热点候选序列都不足以单条序列解决整个植物界的物种鉴定问题,结合本次关于matK、rbcL序列的研究结果与陈士林等人,姚辉等人关于ITS2的研究结果,认为在植物条形码研究中可以针对不同类群选择ITS2、matK、rbcL序列其一或联合,进行植物DNA条形码研究工作。
Objective:To find a universal DNA barcode sequence for plant species comparing with the candidate plant barcode sequences.
Methods:490 samples of 223 species from 98 genus of the family Araceae and 300 samples of 192 species from 72 genera of the family Rutaceae were tested for identification efficiencies of some hot regains as the plant barcodes. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence and barcoding gaps were assessed. After obtaining the desired results, we tested the identification efficiency of matK and rbcL in more than 27,000 samples.
Results:matK sequence performed best in the all regions in the family Araceae.Then we extended to the whole vascular plants and found that the identification rate of matK was 70.1% in all 15765 samples. While 70.2% genus had an identification rate no less than 70% in all 1674 observed genus (genus containing no less than two species), and 12.7% genus has an identification rate less than 40%.As for rbcL, the identification rate is 61.6% in whole 11257 samples, while 56.4% genus had an identification rate no less than 70% in all 1619 observed genus (genus containing no less than two species) and 21.6% genus has an identification rate less than 40%. Because of the defect of matK and rbcL sequence, we continued to select barcode sequences using the barcode samples in the family Rutaceae. We found that the ITS2 locus had the highest identification efficiency among all tested regions. In a previous study, we found that ITS2 was able to discriminate a wide range of plant taxa, and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore, we propose that ITS2 is a promising candidate barcode for plant species identification.
Conclusion:Compared to CO1 which was suitable to study DNA barcoding in animals, there is no single sequence suitable for identification of species throughout the plant kingdom. Based on our results and previous studies, we recommend the 3-locus combination of ITS2+matK+rbcL as the plant barcode. This core 3-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.
方法：选取天南星科98属223种490个样本和芸香科72属192种300个样本,针对目前热点条形码序列进行验证,考察的指标为样本的PCR扩增效率；候选序列种间、种内变异情况的6个“阈值”、种间和种内变异的"barcoding gap"以及"BLAST 1法”和“最近距离法”测得的种、属水平的鉴定成功率。并将rbcL、matK序列扩大到整个维管植物超过27000个样本,在更广泛的类群更多近缘物种存在时判断各自的鉴定能力。
结果：在天南星样本的各项考察指标中matK序列表现最为优异,进而扩大到维管植物中考察发现,matK序列在全体15765样本的鉴定效率为70.1%,而在考察的1674个属(属内物种数≥2)中,72.7%的属鉴定效率≥70%,12.7%的属鉴定效率≤40%;rbcL序列在全体11257样本的鉴定效率为61.6%,而在考察的1619个属(属内物种数≥2)中,56.4%的属鉴定效率≥70%,21.2%的属鉴定效率≤40%。由于matK、rbcL序列存在缺陷,我们在芸香科中对比陈士林等人的推荐ITS2和目前的热点条形码序列,发现ITS2序列在芸香科大量近缘属种存在时各考察指标则优于其他序列,同时基于课题组姚辉等人关于ITS2在大样本量的研究结果发现ITS2序列能较好的鉴定matK+rbcL复合序列无法鉴定的属。
结论：相对于CO1基因在动物条形码研究中的优异表现,植物条形码研究中目前所有的热点候选序列都不足以单条序列解决整个植物界的物种鉴定问题,结合本次关于matK、rbcL序列的研究结果与陈士林等人,姚辉等人关于ITS2的研究结果,认为在植物条形码研究中可以针对不同类群选择ITS2、matK、rbcL序列其一或联合,进行植物DNA条形码研究工作。
Objective:To find a universal DNA barcode sequence for plant species comparing with the candidate plant barcode sequences.
Methods:490 samples of 223 species from 98 genus of the family Araceae and 300 samples of 192 species from 72 genera of the family Rutaceae were tested for identification efficiencies of some hot regains as the plant barcodes. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence and barcoding gaps were assessed. After obtaining the desired results, we tested the identification efficiency of matK and rbcL in more than 27,000 samples.
Results:matK sequence performed best in the all regions in the family Araceae.Then we extended to the whole vascular plants and found that the identification rate of matK was 70.1% in all 15765 samples. While 70.2% genus had an identification rate no less than 70% in all 1674 observed genus (genus containing no less than two species), and 12.7% genus has an identification rate less than 40%.As for rbcL, the identification rate is 61.6% in whole 11257 samples, while 56.4% genus had an identification rate no less than 70% in all 1619 observed genus (genus containing no less than two species) and 21.6% genus has an identification rate less than 40%. Because of the defect of matK and rbcL sequence, we continued to select barcode sequences using the barcode samples in the family Rutaceae. We found that the ITS2 locus had the highest identification efficiency among all tested regions. In a previous study, we found that ITS2 was able to discriminate a wide range of plant taxa, and here we confirmed that ITS2 was also able to discriminate a number of closely related species.Therefore, we propose that ITS2 is a promising candidate barcode for plant species identification.
Conclusion:Compared to CO1 which was suitable to study DNA barcoding in animals, there is no single sequence suitable for identification of species throughout the plant kingdom. Based on our results and previous studies, we recommend the 3-locus combination of ITS2+matK+rbcL as the plant barcode. This core 3-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.
引文
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[2]曹琳,刘忠权,郝明干,等.不同种类中药材的DNA提取方法[J].中国现代应用药学杂志,2004,21(6)：465-468.
[3]Muller P, Jesnowski R, Liebe S,et al.Simple method for DNA extraction from pancreatic juice for PCR amplification assays.[J].Int J Pancreatol,1999,25(1):39-43.
[4]易庆平.柿属植物DNA提取纯化检测技术体系的建立[D].华中农业大学,2006.
[5]Kress W, Wurdack K, Zimmer E.Use of DNA barcodes to identify flowering plants[J].Proc Nat Acad Sci USA,2005,102:8369-8374.
[6]王培训,黄丰,周联.植物中总DNA提取方法的比较[J].中药新药与临床药理,1999,10(1)：18-20.
[7]郭宝林,李家实,阎玉凝,等.中药材DNA分子标记研究的技术问题Ⅰ植物药基因组DNA的提取[J].中草药,2000,31(12)：951-952.
[8]蔡世珍,邹忠梅,徐丽珍,等.半夏属药用植物的研究进展[J].国外医学中医中药分册,2004,26(1)：17-24.
[9]于强,于洋.天南星化学成分和药理作用研究概况[J].中医药信息,2007,13(2)：56-59
[10]孙桂波,张小虎.石菖蒲的药理作用研究集要[J].中医药学刊,2002,4(5)：13-17
[11]Rao S K, Kumar S K, Biswas J, et al.Self-induced corneal crystals:a case report.[J].Cornea,2000,19(3):410-411.
[12]Hebert P D, Cywinska, Ball.Biological identifications through DNA barcodes[J].Proc R Soc Lond B Biol Sci,2003,270:313-321.
[13]Gregory T. DNA barcoding does not compete with taxonomy[J]. Nature,2005,434:1067.
[14]Miller S E.DNA barcoding and the renaissance of taxonomy[J]. Proc Natl Acad Sci U S A,2007,104:4775-4776.
[15]Hebert, Ratnasingham, Dewaard. Barcoding animal life:cytochrome c oxidase subunit 1 divergences among closely related species[J].Proc R Soc Biol Sci Ser B,2004,270:S96-S99.
[16]Vences, Thomas, Bonett. Deciphering amphibian diversity through DNA barcoding:chances and challenges[J].Philos Trans R Soc Lond B Biol Sci,2003,360:1859-1868.
[17]Ward, Zemlak, Innes.DNA barcoding Australia's fish species[J]. Philos Trans R Soc Lond B Biol Sci,2005,360:1847-1857.
[18]Janzen, Hajibabaei, Burns.Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding[J].Philos Trans R Soc Lond B Biol Sci,2005(360):1835-1845.
[19]Chase, Cowan, Hollingsworth. A proposal for a standardised protocol to barcode all land plants[J].Taxon,2007,56:295-299.
[20]Kress, Erickson. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region[J].PLoS ONE,2007,2:e508.
[21]Pennisi E.Wanted:a barcode for plants[J].Science,2007,318: 190-191.
[22]CBOL P W G. A DNA Barcode For Land Plants.[J].Proc Natl Acad Sci USA,2009,106:12794-12797.
[23]Meyer C P, Paulay G. DNA barcoding:error rates based on comprehensive sampling[J].PLoS Biol,2005,3:e422.
[24]Chen S L, Yao H, Han J P, et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PLoS ONE,2010,5:e8613.
[25]Ross H A, Murugan S,Li W L. Testing the reliability of genetic methods of species identification via simulation[J].Syst Biol,2008,57: 216-230.
[26]Hollingsworth P.DNA barcoding plants in biodiversity hot spots: Progress and outstanding questions[J].Heredity,2008,101:1-2.
[27]Sass C,Little D, Stevenson D, et al. DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads.[J].PloS One,2007,2:e1154.
[28]Newmaster S,Fazekas A, Steeves R. Testing candidate plant barcode regions in the Myristicaceae[J].Molecular Ecology Resources,2008,8: 480-490.
[29]Fazekas A, Burgess K, Kesanakurti P.Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well[J].PloS One,2008,3:e2802.
[30]Lahaye R, Bogarin D. DNA barcoding the floras of biodiversity hotspots[J].Proc Natl Acad Sci USA,2008,105:2923-2928.
[31]Newmaster, Fazekas, Ragupathy. DNA barcoding in land plants: evaluation of rbcL in a multigene tiered approach[J].Can J Bot,2006,84: 335-341.
[32]Erickson D, Spouge J, Resch A. DNA barcoding in land plants: developing standards to quantify and maximize success[J].Taxon,2008, 57:1304-1316.
[33]朱英杰,陈士林,姚辉.重楼属药用植物DNA条形码鉴定研究[J].药学学报,2010,45：376-385.
[34]Song J, Yao H, Li Y. Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique.J Ethnopharmacol[J].124,2009:434-439.
[35]Keller A,Schleicher T,Schultz J.5.8S-28S rRNA interaction and HMM-based ITS2 annotation[J].Gene,2009,430:50-57.
[36]Gonzalez M, Baraloto C,Enge J. Identification of Amazonian trees with DNA barcodes[J].PLoS One,2009,4:e7483.
[37]Chase M, Salamin N, Wilkinson M. Land plants and DNA barcodes: short-term and long-term goals[J].Philos Trans R Soc Lond B Biol Sci, 2005,360:1889-1895.
[38]Miao M, Warren A, Song W. Analysis of the internal transcribed spacer 2 (ITS2) region of scuticociliates and related taxa (Ciliophora, Oligohymenophorea) to infer their evolution and phylogeny[J].Protist, 2008,159:519-533.
[39]Muller T, Philippi N, Dandekar T. Distinguishing species[J].RNA, 2007,13:1469-1472.
[40]Shaw J, Lickey E, Beck J. The tortoise and the hare. II. Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis[J].Am J Bot,2005,92:142-166.
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