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Herbal ethnomedicine constituents, being unstructured, are difficult to identify at species level by appearance. Several studies of plant DNA barcoding have generated a huge number of reference sequences in the database (NCBI) for several authentically identified plant species. We tested identification of herbal constituents at the species level in a few ethnomedicine based on the matK sequences and following analysis through Basic Local Alignment Search Tool. Fresh leaves and herbal juices of different ethnomedicine samples were collected from the herbalists and recorded by the common vernacular name. PCR products for matK barcodes (585 bp - 872 bp) were recovered using a single set of primer and sequenced. The denovo sequences showed high similarity (99%100%) with the conspecific sequences in the database. Therefore, different herbal constituents were readily identified, except a single case, at the lowermost taxonomic level based on matK sequence and following the DNA barcoding technique, indicating the novelty of matK in ethnobotany research. Keywords Ethnomedicine · Herbal juice · DNA barcoding · matK · Sequence homology Received 24 September 2012 Accepted 21 December 2012 Pradosh Mahadani, Sankar K Ghosh* DNA barcode and Genomics Laboratory, Department of Biotechnology, Assam University, Silchar-788011, Assam, India © Versita Sp. z o.o. Introduction The concept of DNA barcoding has become very popular for species level identification and is based on the species-specific variations between the short DNA sequences from a uniform locality of the genome [1]. In 2009, CBOL Plant Working Group proposed to use defined portions of plastid gene rbcL and matK as standard DNA barcode for plants and to be supplemented with additional regions as required [2]. But, the low discriminating power of rbcL gene has been reported [3]. The goal of DNA barcoding is to distinguish the majority of world species by using one or a few regions of DNA sequence and to produce a large scale reference sequence library of life on the earth. Several studies of plant DNA barcoding have generated a huge number of reference DNA barcode sequences from taxonomically authenticated species [2-7]. Therefore, an approach of similarity search with reference database would be potential to identify species from any unstructured plant part. The traditional knowledge of using plants for treatment of various ailments in Northeast India is rich due to high diversity of tribes as well as rich diversity of plants [8]. Such rich knowledge is least explored and remained fragmentary. Moreover, there is a trend of not revealing the knowledge by herbal medicine provider to common people. Applications and efficacies of the herbal medicine depend critically on the accuracy in identifying the source plant. Appearance and the conventional morphological identification system do not easily lead to identify the constituent species in juice and powder [9]. Nevertheless, substitutes and adulterants, for profit making, are also in practice that undermined the quality of ethnomedicine. This study was carried to test the application efficiency of matK for species level identification of important ethnomedicinal constituents. We generated matK sequences from some well known, commonly available and valuable ethnomedicine, and compared with public reference database. Methods Fresh leaves (100g) and leaf juices (200ml) of different ethnomedicine samples were purchased from herbalists. As leaves and juices were unidentifiable by appearance, we recorded the common vernacular name in local language (Bengali) and assigned the sample ID to each material (Table 1). Some of the traditional uses of the study samples are like Aloe vera juice is taken for curing digestion and inflammation problem, Cajanus cajan for curing jaundice; Cynodon dactylon for dysentery, Hibiscus rosa-sinensis (leaves in the form of paste) as anti-inflammatory agent, Senna hirsuta and Senna obtusifolia independently for curing skin diseases, and Acmella oppositifolia is used in cuts as antiseptic [10]. Here, we were avoiding the admixture sample. * E-mail: drsankarghosh@gmail.com P. Mahadani, S.K Ghosh Table 1. List of sample ID and sample type with their common and scientific name (according to the Flora of Assam), Accession Number (NCBI) and Length of sequences are also given. Sample ID AUMP16 AUMP28 AUMP21 AUMP2 AUMP4 AUMP61 AUMP63 Sample type Leaf juice Juice Leaf juice Leaf juice Young Leaf Young Leaf Young Leaf Common Name in Bengali Ghrita kumari Durba ghash Akarkara Arhar Jaba Swarnapatri Chakunda Scientific Name (according to Flora of Assam) Aloe vera Cynodon dactylon Acmella oppositifolia Cajanus cajan Hibiscus rosa-sinensis Senna hirsuta Senna obtusifolia Accession No JN228939 JN228941 JN228937 JN228940 JN228942 JQ582660 JQ582661 matK Sequence Length (bp) 781 872 823 771 585 639 634 40mg wet fresh leaves were homogenized in DNA extraction buffer (50mM Tris HCl pH 8.0, 25mM EDTA pH 8.0, and 150mM NaCl, 2µl/ml - mercaptoethanol). In case of juice, 3ml sample was centrifuged @14000x g for 1 min and supernatant was discarded [11]. 1ml of DNA extraction buffer and more 20µl/ml - mercaptoethanol was added into precipitation and incubated in a water bath at 65° C for one hour. Genomic DNA was extracted in less than one hour using Potassium acetate (5M, pH 9.0), Phenol: Chloroform: Isoamyl alcohol (25:24:1), Chloroform: Isoamyl alcohol (24:1). PCR was performed using primer matK X F 5'-TAATTTACGATCAATTCATTC-3' and matK 5r 5'-GTTCTAGCACAAGAAAGTCG-3' [12]. The PCR mixture contained 20ng genomic DNA, 0.2mM of each dNTPs, 50 pmole of each primer, 0.5 units of high fidelity Taq polymerase enzyme (4328212, Applied Biosystem), 1x buffer and 1.5mM MgCl2. PCR in a reaction mixture of 30µl was prepared with the PCR thermal profile as 94º C for 3 min, 30 cycles at 94º C for 45 sec; 48º C for 45 sec; 72º C for 45 sec and a final extension at 72º C for 10 min. The PCR product was checked by 1.5% agarose gel electrophoresis. The PCR products of expected size were extracted using QIA quick PCR purification kit (QIAGEN, Cat. No. 28704). The purified PCR products were sequenced bidirectionally using automated DNA sequencer (ABI 3700). Trace files were assembled in Applied Biosystem Sequence Scanner v1.0 and sequences with greater than 2% ambiguous bases were discarded using QV of 40 for bidirectional reads. Manual editing of raw traces and subsequent alignments of forward and reverse sequences enabled us to assign edited sequences for most species. The 3' and 5' terminals were clipped to generate consensus sequences for each sample. GenBank database was searched using megablast during NovemberDecember 2011 with default parameter adjusted to retrieving 5000 sequences. In most of the case, this corresponded to the sequence with the high BLAST score. In other cases, the closest match was a shorter target with a higher percent identity. Ambiguous bases in target sequence were considered as matching. A similar procedure was followed for BOLD searches. But, BOLD was less well populated with plant DNA barcode sequences used in ethnomedicine. So, subsequent analysis was not performed in BOLD. Results and Discussion Successful amplification was achieved using a single set of primer for the enough length of readable matK barcode sequences (585 bp 872 bp) from selected ethnomedicinal juices and leaves. The BLAST searches by each sample sequence in GenBank revealed the closest matches with the same species and nearest neighbour (NN) of same or different genus. matK sequence of sample AUMP16 showed 100% identity with Aloe vera and 99% with both Aloe compressa and Aloe capitata. matK sequence of sample AUMP28 showed 99.9% similarity with Cynodon dactylon and 99% with Cynodon transvaalensis and Brachyachne ciliaris. The samples AUMP61, AUMP21 and AUMP2 showed 99% identity with Senna hirsuta, Acmella oppositifolia and Cajanus cajan respectively while a below 97% NN similarity with same or different genus was also recorded. AUMP63 showed 100% identity with Senna obtusifolia and 98% NN identity with Senna alata. However, the sample AUMP4 showed 100% identity simultaneously with Helicteropsis microsiphon and Hibiscus rosa-sinensis and remained inconclusive (Figure 1). We authenticated the species name not only based on BLAST result but also by cross checking the common vernacular name as per the Flora of Assam [13] (Table 1). Sample AUMP4 matK sequence was closest to both Helicteropsis microsiphon and Hibiscus rosa-sinensis. It may be due to short sequence length (585 bp) in comparison to other barcode sequences. Therefore, according to vernacular name, the sample AUMP4 was submitted to GenBank as Hibiscus rosa-sinensis (Table 1). The accuracy of DNA barcoding for species identification relies on sufficient sequence difference between closely related species. However, recognition of an ideal plant DNA barcode locus also largely depends on universality, sequence quality and coverage [2]. Plant genera with possible occurrence of natural hybridization and gene introgression may be quite challenging in search of universal loci for plant DNA barcode [14,15]. Plant DNA barcoding in these cases may be problematic and demanding. Longer sequence or additional barcode markers could be provide a better resolution in case of Hibiscus rosasinensis. Large sample sizes were required to increase the power of the test with such members, but the limited number Figure 1. Species identification based on matK barcode. For each sample, sample ID, graphical representation of similarity search result (BLAST) are shown. Color bars depict percentage identity to close match with same species and Nearest Neighbor (NN) in the same or different genus, with scale at bottom. of matK sequences in the database posed a limitation to flag diagnostic nucleotide positions. The matK gene showed the relatively small number of position's differences that distinguish many closely related plant species by comparing the closest match with identical species and NN of same or different genus [16]. Single loci matK showed enough discrimination power among the studied medicinal plant species which reckoned the other findings [6,7,17]. Therefore, DNA barcoding, using matK gene as a potential marker, can be adopted for studying and identifying medicinal plant products that are unidentifiable by morphology alone and for detecting fraudulence that would help in developing ethnobotany research. Acknowledgement We acknowledge the Department of Biotechnology, Govt. of India for the infrastructural support and UGC - JRF in Engineering & Technology fellowships to the author (PM). We are also thankful to Dr. Boni Amin Laskar (DBT-RA fellow) for critical comments and editing of the manuscript.
DNA Barcodes – de Gruyter
Published: Feb 14, 2013
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