Use of DNA barcode sensu lato to identify traditional Tibetan medicinal plant Gentianopsis paludosa (Gentianaceae)

Authors

  • Chun-Ying XUE,

    Corresponding author
    1. (Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China)
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  • De-Zhu LI

    1. (Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China)
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 Author for correspondence. E-mail: chyxue@mail.kib.ac.cn; Tel.: 86-871-5223508; Fax: 86-871-5217791.

Abstract

Abstract Gentianopsis paludosa (Hook. f.) Ma (Gentianaceae) is an important species in Tibetan folk medicine commonly used to clear away the “heat evils” and toxic materials. A survey of market samples revealed that nine adulterant species, Gentianopsis barbata, G. contorta, G. grandis, Halenia elliptica, Lomatogonium macranthum, L. rotatum, Swertia angustifolia, S. bifolia and S. erythrosticta, are often marketed as G. paludosa. Methods to distinguish G. paludosa from its adulterants are limited by current morphological and chemical methods. DNA sequence analysis of the nuclear ribosomal DNA internal transcribed spacer region (ITS) was used in the differentiation of G. paludosa from the adulterant species. The data showed that the internal transcribed spacer regions differ significantly between G. paludosa and all nine adulterant species, so that they could be easily distinguished at the DNA level.

Gentianopsis paludosa (Hook. f.) Ma (Gentianaceae), also known as “shi sheng bian lei”, is an annually growing herb in bosk, meadow, and damp hillsides at an altitude of 2500–4500 m. The plant is distributed in China, India, Nepal, and Bhutan (Ho et al., 1988). In China, it grows in the northwest region and is called “ji he di” by local people (Yang, 1991). The whole herb of G. paludosa has long been used in traditional Tibetan folk medicine for benefiting the liver, clearing away the “heat evils” and toxic materials (Guo, 1987; Yang, 1991). Its main function in indigenous medicine is for the treatment of conjunctivitis, hypertension, haemorrhoids, hepatitis, nephritis, gastroenteritis, dyspepsia, fever, influenza, and diarrhoea (Guo, 1987). Phytochemical studies showed that G. paludosa contained xanthones, terpenoids, and flavonoids (Zhang et al., 1980; Wang et al., 2004, 2005). However, according to our investigation, there are at least nine adulterants on the market bearing the “shi sheng bian lei” commodity name: Gentianopsis barbata (Froel.) Ma, Gentianopsis contorta (Royle) Ma, Gentianopsis grandis (H. Smith) Ma, Halenia elliptica D. Don, Lomatogonium macranthum (Diels & Gilg) Fern., Lomatogonium rotatum (L.) Fries ex Nym., Swertia angustifolia Buch.-Ham ex D. Don, Swertia bifolia Batal., and Swertia erythrosticta Maxim. This widespread adulteration is causing serious confusion in the identification and variation in the quality of this medicinal material. Although these species differ in quality and effect, they share similar morphology and anatomy, for example, the inflorescence patterns and flower features (Ho et al., 1988). Identification of plant at the species level is traditionally achieved by careful examination of the specimen's macroscopic and microscopic morphology. This work usually needs to be carried out by a specially trained expert. Furthermore, many commercial products are sold either in dried forms or in processed material, rendering their authentication by morphological methods very difficult, if not impossible. Therefore, it is very difficult to distinguish G. paludosa from these adulterant species using morphological or histological characteristics.

DNA barcoding, taxon identification using a standardized DNA region, has received much attention recently. Based on different priorities given to the criteria used for designing the molecular markers, DNA barcoding has been derived from two approaches, DNA barcoding sensu stricto and DNA barcoding sensu lato. DNA barcoding sensu stricto corresponds to the identification of the species level using a standardized DNA fragment. The marker should be variable, standardized, and phylogenetically informative. This definition fit with the view of the Consortium for the Barcode of Life (CBOL, 2009). The definition of DNA barcoding sensu lato is much less restrictive. It corresponds to the identification of any taxonomic level using any DNA fragment. The marker should be extremely robust and short (Taberlet et al., 2007). DNA barcoding sensu lato, or DNA-based taxon identification using diverse techniques, can be useful for scientists from special fields, such as traditional Chinese medicine, forensic science, and food industries.

In this study, the internal transcribed spacer (ITS) regions of G. paludosa and its nine adulterant species were sequenced and compared to explore the possibility of using them to differentiate these species.

1 Material and methods

1.1 Source of samples

Gentianopsis paludosa and nine adulterants were collected in Qinghai and Yunnan provinces of China. Table 1 lists all taxa included in this study, together with voucher information and GenBank accession numbers for ITS sequences. The plant specimens in this study were identified by the authors, and voucher specimens were deposited in the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN).

Table 1.  List of plant material used in this study
TaxonVoucherGenBank accession numbers
Gentianopsis barbata (Froel.) MaXCHY 2003108AF346007
G. contorta (Royle) MaXCHY 2003123AJ318539 AJ410318
G. grandis (H. Smith) MaXCHY 2001018DQ317511 DQ317512
G. paludosa (Hook. f.) MaXCHY 2001019Z48106 Z48131
Halenia elliptica D. DonXCHY 2003005AF346012
Lomatogonium macranthum (Diels & Gilg) Fern.XCHY 2003045AF346011
L. rotatum (L.) Fries ex NymXCHY 2003056AF346010
Swertia angustifolia Buch.-Ham ex D. DonXCHY 2001013AJ318551 AJ410330
S. bifolia Batal.Exp. Qingzang 705DQ317490
S. erythrosticta MaximXCHY 2001003AF255914

1.2 DNA extraction, polymerase chain reaction amplification and sequencing

DNAs were extracted using the CTAB method of Doyle & Doyle (Doyle & Doyle, 1987). For the whole ITS region amplification, the primers ITS4 and ITS5 were used (White et al., 1990). The polymerase chain reaction (PCR) reaction volumes (20 μL) contained 1.5 U of AmpliTaq DNA polymerase (TaKaRa Biotechnology, Dalian, China), and the amplification reactions were carried out in a GeneAmp 9600 thermal cycler (Perkin-Elmer 9600; Applied Biosystems, Foster City, CA, USA). The basic PCR programs were 3 min at 97°C, followed by 35 cycles of 45 s at 94°C, 30 s at 55°C, and 45 s at 72°C, with a final elongation of 7 min at 72°C. Double-stranded products were purified using a Watson (Shanghai, China) PCR Purification Kit. Sequencing reactions were carried out using an ABI PRISM Dye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems).The products of the sequencing reaction were electrophoretically separated on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Each sample was sequenced in both directions, and the sequencing was repeated three times.

1.3 Sequence analysis

The DNA sequences were compared and aligned using the programs MegAlign (DNAStar) (http://www.dnastar.com/t-sub-products-lasergene-megalign.aspx) and Mega 3.1 (http://www.megasoftware.net), and further verified by comparison with the sequences of other species in the genus Gentianopsis using a blast search at the National Center for Biotechnology Information website (http://www.nibi.nlm.nih.gov).

2 Results

The length of the ITS (ITS1 and ITS2) sequences in the 10 species ranged from 453 to 459 bp, giving an aligned matrix of 465 characters (Fig. 1). Of these, 330 sites were constant, 72 were variable, and 63 (19.10%) were parsimony informative. Gentianopsis paludosa had a 454-base sequence, and the percentage of G + C was 59.0% (Table 2). The pairwise distances between samples were estimated. The nucleotide sequence divergence between different populations of the same species was zero for G. paludosa, clearly showing that the ITS sequences of G. paludosa are homologous regardless of its geographical origin. Gentianopsis paludosa displayed sequence divergences at 73–58 positions from the six non-Gentianopsis species and 19–30 positions from the four Gentianopsis species. There is a much larger sequences divergence of 73 positions between G. paludosa and Halenia elliptica, and a small one of 19 positions between G. paludosa and G. barbata. Within the genus Gentianopsis, there is a small sequence divergence of 12 positions between G. barbata and G. grandis, and a much larger one of 30 positions between G. grandis and G. contorta (Table 3). The inter-species nucleotide sequence divergence between G. paludosa and the nine adulterant species therefore ranged from 19 to 73 positions, which is much larger than the intra-species variation of zero. On this basis the ITS (ITS1 and ITS2) region of G. paludosa could be adopted as a molecular marker for the accurate identification of this species from the adulterant species.

Figure 1.

Aligned sequences of ribosomal DNA internal transcribed spacers from Gentianopsis paludosa and nine adulterant species in the region. Dashes are gaps required for alignment.

Table 2.  Sequence characteristics of the internal transcribed spacer (ITS) regions of the studied species
ComparisonITS
Length range (bp)453–459
Aligned length (bp)465
G+C content range (%)57.77–61.66
G+C content mean (%)59.89
Sequence divergence (%)0.9–16.7
Number of variable sites (%)72
Number of constant sites (%)330
Number of informative sites (%)63
Table 3.  Pairwise distances between the studied species based on internal transcribed spacer regions
 12345678910
  1. Total character differences are below diagonal; mean character differences are above diagonal. —, Same sample has no character differences.

1 Gentianopsis paludosa0.0690.0440.0460.1350.1330.1330.1560.1510.167
2 G. contorta300.0530.0600.1280.1310.1260.1580.1540.163
3 G. barbata19230.0280.1060.1030.1100.1260.1220.131
4 G. grandis2026120.1170.1150.1150.1420.1380.151
5 Swertia bifolia595646510.0300.0800.0960.0940.115
6 S. erythrosticta58574550130.0780.0960.0940.117
7 S. angustifolia5855485035340.1060.1060.131
8 Lomatogonium macranthum686955624242460.0090.122
9 L. rotatum6667536041414640.119
10 Halenia elliptica737157665051575352

3 Discussion

The World Wildlife Fund estimates that 80% of the world's population uses traditional medicines for healing and curing diseases (http://www.worldwildlife.org/what/globalmarkets/wildlifetrade/faqs-medicinalplant.html). The increased demand for botanical products is met by an expanding industry and accompanied by calls for assurance of quality, efficacy, and safety (Chen et al., 2010). Plants used as drugs, dietary supplements, and herbal medicines are identified at the species level. Unequivocal identification is a critical step at the beginning of an extensive process of quality assurance and is important for the characterization of genetic diversity, phylogeny, and phylogeography as well as the protection of endangered species (Sucher & Carles, 2008).

Using DNA-based methods, species identification has been achieved using DNA that was isolated from fresh and dried plant parts, plant extracts, processed herbal drugs, as well as finished products such as herbal teas, tablets, and capsules. In addition, DNA barcodes will be a useful and powerful tool for non-professional users such as customs officers, traditional drug producers and managers, and forensic specialists. DNA-based methods can be useful in quickly and efficiently pinpointing adulterated or misidentified raw materials, which can then be discarded without further need for time- and resource-consuming morphological, physical, and phytochemical examinations (Lau & Shaw, 2001; Ding et al., 2002).

The length of the ITS in Gentianopsis paludosa is short (456 bp), which advantages the amplification of degraded DNA. The inter-species nucleotide sequence divergence between G. paludosa and the nine adulterant species studied ranged from 19 to 73 positions, which is much larger than the intra-species variation of zero. The inter-specific divergence at ITS is higher than the intra-species divergence. Therefore, the ITS region could be potentially used to identify medicinal plant G. paludosa and its closely related species. This study agrees that with the exception of 5.8S, the ITS of nuclear ribosomal DNA and regions of the ITS could be potential barcodes (Chase et al., 2005; Kress et al., 2005).

Acknowledgments

Acknowledgements  Support for this research was provided by the National Natural Science Foundation of China (Project 30770153 to CY Xue) and the Large-Scale Scientific Facilities of Chinese Academy of Sciences (Grant No. 2009-LSF-GBOWS-01).

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