Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

PLOS ONE, Jan 2010

Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.

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Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

et al. (2010) Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLoS ONE 5(1): e8613. doi:10.1371/journal.pone.0008613 Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species Shilin Chen 0 Hui Yao 0 Jianping Han 0 Chang Liu 0 Jingyuan Song 0 Linchun Shi 0 Yingjie Zhu 0 Xinye Ma 0 Ting Gao 0 Xiaohui Pang 0 Kun Luo 0 Ying Li 0 Xiwen Li 0 Xiaocheng Jia 0 Yulin Lin 0 Christine Leon 0 M. Thomas P. Gilbert, Niels Bohr Institute and Biological Institutes, Denmark 0 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College , Beijing , People's Republic of China, 2 Li Ka Shing Faculty of Medicine, The University of Hong Kong , Hong Kong , People's Republic of China, 3 Department of Pharmacy, Hubei University of Chinese Medicine , Wuhan, Hubei, People's Republic of China, 4 Royal Botanic Gardens, Kew, Richmond , United Kingdom Background: The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings: Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions: The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa. - Funding: This work is supported by the International Cooperation Program of Science and Technology (No. 2007DFA30990) and the Special Foundation of Ministry of Health (No. 200802043) granted to S.L.C. This work is also supported by grants from the Hong Kong Research Grant Council (HKU 7526/06M) to C.L. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The World Health Organization estimates that 80 percent of the worlds population utilizes traditional medicines for healing and curing diseases (http://www.worldwildlife.org/what/globalmarkets/ wildlifetrade/faqs-medicinalplant.html). There is an increasing international market for medicinal plants, which are used both for herbal medicine and for pharmaceutical products. Medicinal plants cover a wide range of plant taxa and closely related species. According to surveys in China, medicinal plants belong to 11,146 species from 2,309 genera of 383 families, representing a rich biodiversity. Accurate and rapid authentication of these plants and their adulterants is difficult to achieve at the scale of international trade in medicinal plants. We aim to provide a practical and powerful tool for identifying medicinal plants and their adulterants in trade and for ensuring safety in their use. The term DNA barcode for global species identification was first coined by Hebert in 2003 [1,2] and has gained worldwide attention in the scientific community [37]. Recognition of animals, plants and fungi has been performed using this technique [812]. Most researchers agree that the mitochondrial gene encoding cytochrome c oxidase subunit 1 is a favorable region for use as a DNA barcode in most animal species and even in some fungal species, including those of the groups Ascomycota, Basidiomycota and Chytridiomycota. However, the CO1 gene and other mitochondrial genes from plants have limited usefulness for identifying plant species across a wide range of taxa due to the low amounts of variation in the genes, as well as the variable structure of the mitochondrial genome [9,1316]. Thus, screening for single or multiple regions appropriate for DNA barcoding studies in nuclear and plastid genomes in plants has been an important research focus (Fig. 1). First, most single-copy genes in the nuclear genome, as well as their introns, have been excluded as barcode candidates because of the lack of universal primers for their amplification [9]. However, with the exception of 5.8S, the internal transcribed spacer (ITS) (...truncated)


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Shilin Chen, Hui Yao, Jianping Han, Chang Liu, Jingyuan Song, Linchun Shi, Yingjie Zhu, Xinye Ma, Ting Gao, Xiaohui Pang, Kun Luo, Ying Li, Xiwen Li, Xiaocheng Jia, Yulin Lin, Christine Leon. Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species, PLOS ONE, 2010, Volume 5, Issue 1, DOI: 10.1371/journal.pone.0008613