Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2

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Abstract

Aim of the study

To test whether the ITS2 region is an effective marker for use in authenticating of the family Fabaceae which contains many important medicinal plants.

Materials and methods

The ITS2 regions of 114 samples in Fabaceae were amplified. Sequence assembly was assembled by CodonCode Aligner V3.0. In combination with sequences from public database, the sequences were aligned by Clustal W, and genetic distances were computed using MEGA V4.0. The intra- vs. inter-specific variations were assessed by six metrics, wilcoxon two-sample tests and “barcoding gaps”. Species identification was accomplished using TaxonGAP V2.4, BLAST1 and the nearest distance method.

Results

ITS2 sequences had considerable variation at the genus and species level. The intra-specific divergence ranged from 0% to 14.4%, with an average of 1.7%, and the inter-specific divergence ranged from 0% to 63.0%, with an average of 8.6%. Twenty-four species found in the Chinese Pharmacopoeia, along with another 66 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 1507 sequences derived from 1126 species belonging to 196 genera.

Conclusions

Our findings support the notion that ITS2 can be used as an efficient and powerful marker and a potential barcode to distinguish various species in Fabaceae.

Introduction

Fabaceae is the second largest family of medicinal plants, containing over 490 medicinal plant species, most of which have been used as traditional medicines. There are 31 species of medicinal plants belonging to 20 genera in the family Fabaceae that have been described in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2005), as well as numerous species that are included in the Japanese Pharmacopoeia. These species possess important medicinal properties and have been widely used as components of pharmaceutical products. For instance, Glycyrrhiza uralensis, Glycyrrhiza inflata, and Glycyrrhiza glabra, which are all generally used in traditional medicines, have inhibitory effects on HIV replication in vitro and anti-Fas antibody-induced hepatitis (Okamoto, 2000, Watanabe et al., 1996). Trigonella foenum-graecum has been shown to reduce blood glucose levels significantly (Vats et al., 2002). Additionally, plant materials from nearly 290 species belonging to 100 genera of Fabaceae have been reported to be toxic. For example, Acacia rigidula has been shown to contain appreciable levels of toxic alkaloids (Clement et al., 1998), and many of species of the genus Crotalaria contain pyrrolizidine alkaloids, which are toxic to mammals and birds (Williams and Molyneux, 1987).

Commonly used medicinal species and their adulterants are frequently found in the market together. For example, Astragalus membranaceus and Astragalus mongolicus are two of the most popular traditional Chinese medicines. However, Hedysarum polybotrys is often mistaken for Radix Astragali (Ma et al., 2002). Pueraria tuberosa is often used in traditional medicine in India, but there are at least three other botanical entities, Ipomoea mauritiana, Adenia hondala and Cycas circinalis that are traded under the same name (Devaiah and Venkatasubramanian, 2008).

The identification of different species of Fabaceae is difficult when based solely on morphological characteristics (Hou et al., 2008, Newmaster and Ragupathy, 2009); additionally, some limitations in traditional taxonomy prevent this technique from meeting the complicated demands of species recognition (Maddison et al., 2007). As such, a method for the simple and accurate authentication of Fabaceae is indispensible.

Several DNA barcodes (matK, rbcL, psbA-trnH, ITS, rpoC1, etc.) have been developed for the identification of species (Kress and Erickson, 2007, Kress et al., 2009, Lahaye et al., 2008, Song et al., 2009, Newmaster and Ragupathy, 2009, Pang et al., 2010, Luo et al., 2010), but the barcodes for Fabaceae species are limited to a few genera and cannot been applied across the family (Edwards et al., 2008, Hollingsworth et al., 2009, Newmaster and Ragupathy, 2009). Recently, Plant Working Group has recommended rbcL and matK as core DNA barcodes (CBOL Plant Working Group, 2009). However, theoretically, nuclear DNA would provide more information for barcoding than organellar DNA (Chase and Fay, 2009). The effectiveness of ITS and ITS1 have been questioned in regard to improving the quality of primers to enhance their universality (Chase et al., 2007, Kress and Erickson, 2007, Chen et al., 2010). While, being part of ITS, ITS2 is relatively easy to be amplified using one pair of universal primers (Chiou et al., 2007, Chen et al., 2010). In addition, ITS2 has been found to provide taxonomic signatures in systematic evolution (Coleman, 2003, Coleman, 2007, Schultz et al., 2005). The ITS2 region is also a promising potential molecular marker to be used for rapid taxonomic classification (Chiou et al., 2007, Chen et al., 2010).

In the current study, we utilize ITS2 as a DNA barcode to differentiate medicinal plants within the Fabaceae family in order to ensure their safe application in traditional uses.

Section snippets

Plant materials

In our study, 114 samples, which belonged to 85 species from 49 genera, were collected from large geographical areas in China between July 2007 and January 2008 (Online Resource 1). All plant species were identified by Professor Yulin Lin, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Professor Quanru Liu, Beijing Normal University. The voucher samples were deposited in the herbarium of IMPLAD.

We have combined the sequences generated in our lab and

Measurement of DNA divergence for ITS2

Significant variations in DNA sequences between species and smaller variations within species are prerequisites for an ideal DNA marker to use in identifying various species. Therefore, we first characterized the intra- and inter-specific variations in ITS2 sequences. The lengths of the ITS2 sequences used for the analyses ranged from 205 to 249 bps and 185 to 301 bps, in dataset 1 and dataset 2, respectively. In dataset 1, the inter-specific percentages of nucleotide differences ranged from 0% (

Discussion

A rapid and accurate method to authenticate species from the large family Fabaceae is very important to ensure the safe usage of drugs made from these medicinal herbs. To our knowledge, this is the first time that the ITS2 region was used to identify plant materials from Fabaceae with such a large sample size. ITS2 was found to be a sufficiently variable DNA region among Fabaceae species as determination by genetic divergences, and ITS2 also demonstrated a higher capability of successful

Conclusion

In this study, ITS2 was examined for its usefulness in identifying medicinal species of Fabaceae. Our findings show that the ITS2 region can not only be used to identify 24 Fabaceae species in the Chinese Pharmacopoeia, along with another 66 species, including their adulterants, but can also correctly distinguish over 80.0% of species and 100% of genera from the 1507 sequences of Fabaceae species. Hence, ITS2 is a powerful and efficient tool for species identification of medicinal plants and

Acknowledgements

We would like to thank XC Jia for his valuable comments. This work is supported by the International Cooperation Program of Science and Technology (No. 2007DFA30990) and the Special Founding for Ministry of Health (No. 200802043) granted to S.L.C. We also thank the general support by grants from the Hong Kong Research Grant Council (HKU 7526/06M) to C.L.

References (44)

  • M.W. Chase et al.

    Barcoding of plants and fungi

    Science

    (2009)
  • S.L. Chen et al.

    Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species

    PLos One

    (2010)
  • S.J. Chiou et al.

    Authentication of medicinal herbs using PCR-amplified ITS2 with specific primers

    Planta Medica

    (2007)
  • A.W. Coleman

    Pan-eukaryote ITS2 homologies revealed by RNA secondary structure

    Nucleic Acids Research

    (2007)
  • K.M. Devaiah et al.

    Development of SCAR marker for authentication of Pueraria tuberosa (Roxb. ex. Willd.) DC

    Current Science

    (2008)
  • T.T.X. Dong et al.

    Phylogeny of Astragalus in China: molecular evidence from the DNA sequences of 5S rRNA spacer, ITS, and 18S rRNA

    Journal of Agricultural and Food Chemistry

    (2003)
  • S.R Eddy

    Profile hidden Markov models

    Bioinformatics

    (1998)
  • S.R. Eddy

    HMMER: Profile Hidden Markov Models for Biological Sequence Analysis

    (2000)
  • D. Edwards et al.

    DNA barcoding of a large genus, Aspalathus L. (Fabaceae)

    Taxon

    (2008)
  • M.L. Hollingsworth et al.

    Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants

    Molecular Ecology Resources

    (2009)
  • X. Hou et al.

    Molecular phylogeny of Caragana (Fabaceae) in China

    Journal of Systematic Evolution

    (2008)
  • X. Hou et al.

    Inter-specific relationships of Caragana microphylla, C. davazamcii and C. korshinskii (Leguminosae) based on ITS and trnL-F data sets

    Acta Phytotaxonomica Sinica

    (2006)
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