Available online at www.sciencedirect.com GENOMICS PROTEOMICS & BIOINFORMATICS Genomics Proteomics Bioinformatics 10 (2012) 364–367 www.elsevier.com/locate/gpb Application Note DNA Barcode ITS Effectively Distinguishes the Medicinal Plant Boerhavia diffusa from Its Adulterants Dhivya Selvaraj 1, Dhivya Shanmughanandhan 1, Rajeev Kumar Sarma 1, Jijo C. Joseph 1, Ramachandran V. Srinivasan 2, Sathishkumar Ramalingam 1,⇑ 1 Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, India 2 Department of Botany, Bharathiar University, Coimbatore 641046, India Received 2 September 2011; revised 9 March 2012; accepted 21 March 2012 Available online 29 November 2012 Abstract Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant in India and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, a DNA barcoding technique has been applied to identify and distinguish B. diffusa from its closely-related species. The phylogenetic analysis was carried out for the four species of Boerhavia using barcode candidates including nuclear ribosomal DNA regions ITS, ITS1, ITS2 and the chloroplast plastid gene psbA-trnH. Sequence alignment revealed 26% polymorphic sites in ITS, 30% in ITS1, 16% in ITS2 and 6% in psbA-trnH, respectively. Additionally, a phylogenetic tree was constructed for 15 species using ITS sequences which clearly distinguished B. diffusa from the other species. The ITS1 demonstrates a higher transition/transversion ratio, percentage of variation and pairwise distance which differentiate B. diffusa from other species of Boerhavia. Our study revealed that ITS and ITS1 could be used as potential candidate regions for identifying B. diffusa and for authenticating its herbal products. Keywords: Adulterant; Boerhavia diffusa; ITS; DNA barcoding; Punarnava Introduction Boerhavia is one of the highly polymorphic genus in Nyctaginaceae family [1]. About 40 species are distributed in tropical, subtropical and temperate regions. Among these, 6 species are reported in India and Boerhavia diffusa (B. diffusa) is indigenous [2]. B. diffusa is described as Punarnava by an Indian system of medicine, Ayurveda [3]. Roots and whole plants of B. diffusa are used in the Ayurvedic and Unani systems of medicine in Arabian countries [4] and many tribal communities in India still use it for the treatment of jaundice and various other liver disorders. It has anti-inflammatory, diuretic, fibrinolytic, anti-convulsant properties [5–8] and also used as carminatives [9–10]. The two pharmaceutically important alkaloids, Punarna⇑ Corresponding author. E-mail: rsathish@buc.edu.in (Ramalingam S). vine-1 and Punarnavine-2, belonging to the group of quinolizidine were separated from B. diffusa [11–12]. B. diffusa is known to be extensively adulterated with other species like Boerhavia erecta, Boerhavia repanda, Boerhavia coccinea and Boerhavia verticillata. B. diffusa have taxonomical conflicts with B. coccinea, Boerhavia repens, Boerhavia tetranda and Boerhavia albiflora, making it difficult to distinguish from one another [13–14]. The species B. verticillata display similar morphological features and phytochemical properties with B. diffusa, but they differ by their habits [15]. Determination of plant specimens by DNA barcodes will be an effective, reliable and simple pharmacognostic tool to resolve the confusion in morphological identification. Due to different rates of evolution, nuclear ribosomal internal transcribed spacer (ITS) regions have become the routine marker in evolutionary studies at different taxonomic levels [16,17]. There is a report using the chloroplast intergenic spacer psbA-trnH for identifying 1672-0229/$ - see front matter Ó 2012 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Published by Elsevier Ltd and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.gpb.2012.03.002 365 Selvaraj D et al / ITS Distinguishes Boerhavia diffusa from Its Adulterants the Dendrobium species of Chinese pharmacopoeia and psbA-trnH is recommended as an ideal DNA barcode candidate [18]. Recently the sequence variations are used to develop specific markers for the identification and authentication of drugs and herbal formulations [19]. The objective of the present study is to evaluate an ideal barcode candidate for distinguishing and authenticating the species B. diffusa from its common adulterants. Results Genomic DNA was isolated from the species of B. diffusa, B. repanda, B. erecta and B. verticillata and used for PCR amplification of the ITS and psbA-trnH. The obtained sequences were submitted to GenBank. The size and accession number for the gene ITS and psbA-trnH is shown in Table S1. Additionally, ITS sequences from 11 species of Boerhavia were taken from the GenBank (Table S2) and used for sequence alignments. Multiple sequence alignment and pairwise alignment analysis were performed for nuclear ITS and chloroplast psbA-trnH (Figure S1). The ITS region consists of ITS1, 5.8S rDNA and ITS2. The ribosomal sites of 5.8S rRNA and 28S rRNA are highly conserved. The regions ITS1 and ITS2 were compared by multiple sequence alignment, where ITS1 showed more variation than ITS2. Phylogenetic analysis using ITS1 and ITS2 indicated B. diffusa and B. erecta in the same clade while B. verticillata and B. diffusa was shown in the same clade when using psbA-trnH region for phylogenetic analysis (Figure 1). The tree also constructed using ITS region clearly distin- guished the morphologically similar species B. diffusa from the 14 other species of Boerhavia as shown in the Figure 2. We further analyzed the nucleotide variations of ITS and psbA-trnH between different species. Percentage of variation shown in Figure 3 indicated that ITS demonstrated higher inter-specific divergence. The Wilcoxson rank test indicated significant variation between the species for ITS1 when compared to ITS2 and psbA-trnH. BLAST 1 and distance method also indicated that ITS1 showed higher identification percentage at species level (Table 1). Discussion Recent molecular methods like DNA barcoding have been extensively used for species identification, diversity, forensic medicine and ecological studies [20–21]. It also plays an important role in the identification of traditional medicinal herbs. ITS2 has been effectively used in differentiating morphologically similar species like Swartzia grandifolia and Swartzia longicarpa and also in solving the controversial species Caranga rosea and Caranga sinica of the family Fabaceae [22]. Medicinal plant species like the family Polygonaceae [23] and the genus Dendrobium [24] have been identified using ITS2 region. In addition, ITS1 was used to demonstrate that species of Amomum villosum belongs to the family Zingiberaceae [25]. In our study, multiple sequence alignment of ITS1 and ITS2 from four Boerhavia species showed that B. diffusa had a unique basepair variation, which can distinguish it from the other three species, despite the fact that they share many morphological similarities. On the other hand, although psbA-trnH distinguishes some species of Polygonaceae [18], less sequence variation in psbA-trnH was revealed among the four species of Boerhavia. This result is consistent with a previous report that psbA-trnH does not show any variation for closely-related Cycad species [26]. Hence, ITS1 may be a better barcode region for distinB. repens B. diffusa 100 B. coccinea B. gracillima B. anisophylla B. linearifolia 99 66 B. ciliata B. dominii 94 B. intermedia B. lateriflora 100 B. purpurascens B. erecta 99 74 B. spicata B. repanda 100 B. verticillata 84 88 73 53 0.01 Figure 1 Phylogenetic trees of the four Boerhavia species constructed using ITS1, ITS2 and psbA-trnH Phylogenetic trees were constructed by Minimum Evolution method using ITS1, ITS2 and psbA-trnH, respectively, for the four species of Boerhavia, including B. diffusa, B. erecta, B. repanda and B. verticillata. Figure 2 Phylogenetic tree of the 15 Boerhavia species constructed using ITS Phylogenetic tree was constructed by Minimum Evolution method for the 15 species of Boerhavia using ITS region. 366 Genomics Proteomics Bioinformatics 10 (2012) 364–367 Figure 3 Sequence variation of ITS and psbA-trnH between four Boerhavia species Percentage of nucleotide variations between different Boerhavia species, including B. diffusa, B. erecta, B. repanda and B. verticillata, was indicated for ITS (dark gray) and psbA-trnH (light gray), respectively. Table 1 species Validation of ITS1, ITS2 and psbA-trnH from four Boerhavia Gene name Correct identification (%) BLAST 1 ITS1 ITS2 psbA-trnH Distance At genus level At species level At species level 98 99 94 94 92 91 93.2 91.1 76.86 psbA-trnH F 50 -GTTATGCATGAACGTAAGCTC-30 , psbA-trnH R 50 -CGCGCATGGTGGATTCAAATCC-30 , respectively. The forward primer of ITS region from [29] was modified at the position of 6 (T ! G) and 8 (A ! G). The PCR program was as follows, an initial denaturation at 94 °C 5 min, followed by 35 cycles of 94 °C 1 min, 57 °C 30 s, 72 °C 1 min and final extension at 72 °C 10 min. PCR products were resolved by gel electrophoresis, purified and subjected to sequencing. The obtained ITS and psbA-trnH sequences were deposited in the GenBank of NCBI database (GenBank Accessions: HQ386701, HQ386689, HQ386691, HQ386695, HQ386696, HQ407399, HQ386690 and JF423303) (Table S1). Sequences of ITS genes from 11 additional species of Boerhavia were obtained from GenBank (Table S2). Sequence alignment and phylogenetic analysis The DNA sequences were compared and aligned using the programs ClustalW [30] and MULTALIGN (http:// www.multalin.toulouse.inra.fr/multalin/). Further, the DNA sequences were subjected to BLAST (http:// www.ncbi.nlm.nih.gov/blast/blast.cgi) for better identification of sequence at species level. Phylogenetic trees were constructed with the Minimum Evolution method using MEGA 4.0. The intra-specific variation between the species was calculated using MEGA 4.0 [31] and StatsDirect was used to calculate the Wilcoxon signed rank [32]. guishing the species of Boerhavia, although ITS2 has been widely used to distinguish the plant species [27]. This study clearly indicates that DNA barcoding using candidate like ITS1 is a reliable method for differentiating B. diffusa from the other three species, which can also be applied to rapid identification of medicinal plants and their adulterants or substitutes. Authors’ contributions Materials and methods Competing interests Sample collection The authors have declared that no competing interests exist. Four species of Boerhavia (B. diffusa, B. erecta, B. repanda, B. verticillata) were collected from the regions of Western Ghats (one of the hotspots), Coimbatore, India. The species were collected and identified by the taxonomist. DNA extraction, PCR amplification and DNA sequencing Fresh leaves from each plant species were used for isolating total genomic DNA by CTAB method [28]. The ITS and psbA-trnH gene amplifications were performed using Taq DNA polymerase with the primers indicated below. The forward and reverse primers for ITS and psbA-trnH used were ITS F 50 -GGAAGGAGAAGTCGTAACAAGG-30 , ITS R 50 - TCCTCCGCTTATTGATATGC-30 [28] and SR supervised the research. RVS collected and identified the specimens. DS, DS, RKS and JCJ carried out the experimental study. Dhivya Selvaraj prepared the manuscript and SR revised it. All authors read and approved the final manuscript. Acknowledgements Dhivya Selvaraj thanks the University Grant CommissionResearch Fellowship for Meritorious Students (UGCRFMS). Sathishkumar Ramalingam thanks the financial support from UGC (Grant No. 34-272/2008(SR)). 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