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Identification and Genetic Diversity Analysis Of Electronic Journal of Biotechnology 24 (2016) 1–8 Contents lists available at ScienceDirect Electronic Journal of Biotechnology Research article Identification and genetic diversity analysis of Memecylon species using ISSR, RAPD and Gene-based DNA barcoding tools Bharathi Tumkur Ramasetty a, Shrisha Naik Bajpe a, Sampath Kumara Kigga Kadappa a,RameshKumarSainib, Shashibhushan Nittur Basavaraju c, Kini Kukkundoor Ramachandra a, Prakash Harishchandra Sripathy a,⁎ a Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India b Department of Bio-resource and Food Science, College of Life and Environmental Sciences, Konkuk University, Seoul 143-701, Republic of Korea c Department of Crop Physiology, GKVK, Bangalore, India article info abstract Article history: Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification Received 6 April 2016 is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR Accepted 31 August 2016 and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and Available online 14 September 2016 analyze the diversity of five Memecylon species collected from Western Ghats of India. Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Keywords: Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. atpF-atpH In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated Memecylon species ndh into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and psbA-trnH Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap rpoC1 support between each node, indicating that these grouped species are phylogenetically similar. 5s Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential PCR based techniques candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating Molecular phylogenetic analysis genetic diversity since it has high percent polymorphism compared to RAPD marker. Molecular phylogenesis DNA markers This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/). 1. Introduction Memecylon umbellatum and Memecylon edule Roxb, as separate species. However, Brandis [6], Neginhal [10] and Pullaiah et al. [11] The family Melastomataceae consists of about 166 genera and more treated M. umbellatum as a synonym of M. edule. Bhat [12] mentioned than 4000 species distributed worldwide. The genus Memecylon that M. umbellatum and M. edule treated as co-specific in some of the consists of 300–400 species, distributed in the tropical areas of Asia, regional floras [13]. Nomenclature status of most of the Indian Africa and America [1]. Memecylon species have great importance in Memecylon species is not clear (www.plantlist.org)andalso traditional medicine practices in India. In Ayurveda and Siddha system complexity exists in morphological characters and identification. of medicine, several Memecylon species are used to treat skin Therefore, it has become imperative to study the mechanisms of disorders, stomach disorders, herpes, chickenpox, leucorrhoea, species diversification of Memecylon species [14]. polyuria, menorrhagia, dysentery, bacterial infections, inflammations, In recent years, to authenticate a plant, a number of single loci and diabetes and also has antimicrobial, hepatoprotective and antipyretic combined loci have been used. Based on the performance of seven properties [2,3]. plastid DNA regions (atpF-atpH, matK,c rb L, rpoB, rpoC1, psbK-psbI, and There is a taxonomic ambiguity in identification and distinction of psbA-trnH), the Plant Working Group of the Consortium for the Memecylon species viz., Memecylon malabaricum Clarke and Barcode of Life recommended the combination of rbcL and matK as the Memecylon wightii Thwaites [4,5,6,7,8]. Saldanha [7] stressed the need plant barcode [15]. The study on 5s IGS region of different species of for clarification regarding the relationship between Memecylon Vigna subgenus Ceratotropis [16] was found to be phylogenetically amplexicaule var. malabarica and M. wightii. Vivekanandan [9] treated informative to detect intragenic relationship. The chloroplast intergenic spacer psbA-trnH has been recommended as an ideal DNA barcode candidate [17]. rpoC1, ndh and atpF-atpH are used to evaluate ⁎ Corresponding author. E-mail address: [email protected] (P.H. Sripathy). plant phylogeny with low taxonomic variation [18]. DNA markers Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. RAPD and ISSR are used to establish the phylogenetic relationship http://dx.doi.org/10.1016/j.ejbt.2016.09.001 0717-3458/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 B.T. Ramasetty et al. / Electronic Journal of Biotechnology 24 (2016) 1–8 Table 1 List of Memecylon accessions collected for the study and their Morphological characters. Sl Accession Plant name Voucher No Morphological character for identification References for morphological no. code identification 1 Mu1-Mu10 M umbellatum #IOELP0001a-#IOELP0001j Leaves petiolate and ovate-elliptic. The inflorescence is distinctly Saldanha [7] peduncled and branched. 2 Me11 M. edule #IOELP0001h Leaves ovate, acute at both ends and nerves obscure. The Hooker [5] inflorescence is pedunculate and flowers blue. 3 Mm12-Mm20 M. malabaricum # IOELP0003 Branchlets subterete, Leaves subsessile and rounded, cordate at Saldanha [7] #IOELP0003a-#IOELP0004h base. Inflorescence of subsessile clades. 4 Mw21-Mw24 M. wightii # IOELP0004 Branchlets winged or angled. Leaves subsessile and cordate at base. Ramaswamy [8] #IOELP0004a-#IOELP0004c Flowers pedicellate and fascicled on short tubercles at leafless nodes. 5 Mt25-Mt-27 M. talbotianium #IOELP0002 Leaves distinctly petiolate, elliptic, attenuate at the base and the tip Saldanha [7] #IOELP0002a acute or acuminate. Inflorescence unbranched, at most subsessile. #IOELP0002b among Bacopa monnieri [19], Eucalyptus [20] Gaultheria fragrantissima 2.2. Sequence alignment and analysis [21] and to identify the genus/species of the plants at different taxonomic levels. The nuclear 5s and chloroplast psbA-trnH, rpoC1, ndh, atpF-atpH In the current study, nuclear ribosomal 5s and chloroplast sequences of Memecylon species and related genera were obtained psbA-trnH, atpF-atpH, ndh, rpoC1 sequences are used for validation from GenBank (Appendix 1). The Codon Code Aligner 3.6.1 Clustal and identification of Memecylon species of Western Ghats. RAPD and Omega was used to align nuclear and chloroplast DNA sequence data. ISSR are used to understand the genetic diversity within five The different gene datasets were first analyzed with J model test [25] Memecylon species of the Western Ghats namely M. umbellatum using the Akaike information criterion to find the most appropriate Burm., M. malabaricum Clarke, M. wightii Thwaites, M. edule Roxb. and model for DNA substitution. A phylogenetic tree was constructed by M. talbotianum Brandis. the maximum likelihood (ML) in RAxML-HPC2 7.2.8 [26] with a rapid bootstrap analysis using a random starting tree and 100 bootstrap replicates searching for the best maximum-likelihood tree. 2. Materials and methods RAPD, ISSR bands were scored for 27 samples by visual inspection and fragment sizes were estimated with a medium range ruler Memecylon species plant samples were collected from different (Genei). The scored bands ranged from 100–10,000 bp. The presence regions of Western Ghats, Karnataka, India. Identification of the plant or absence of bands was scored as diallelic for each assigned locus species based on their morphological characteristics was confirmed by (1 = band present; 0 = band absent) and compiled into a matrix. plant taxonomist. A total of 27 accessions representing five species RAPD and ISSR data matrix was constructed containing all scorable namely M. umbellatum, M. edule, M. talbotianum, M. malabaricum and bands. The dissimilarity matrix was used to construct a dendrogram M. wightii were collected for this study (Table 1). using the neighbor-joining method (NJ) with 100 bootstrap replicates. These analyses were carried out using the DARwin 5.0.148 [27].The similarities between matrices based on different marker systems were fi 2.1. DNA extraction, PCR ampli cation and DNA sequencing calculated using the standardized Mantel co-efficient [28] using the NTSYSpc ver. 2.01 program [29]. For each primer combination Percent DNA was isolated using CTAB method [22]. The 5s [16], psbA-trnH polymorphisms were calculated [30]. [23], rpoC1 and atpF-atpH [24] and ndh gene amplifications were performed as per the protocol (Table S1). The amplified products were sequenced (Chromous Biotech, Bangalore). 3. Results The sequences were submitted to the GenBank, NCBI (Table 2). The PCR Protocol was optimized by using varying concentrations of The resulted sequences of the 5s, psbA-trnH, rpoC1, ndh and template DNA, dNTPs, Taq DNA polymerase and annealing atpF-atpH were subjected for the BLAST search. The sequences temperature. For RAPD, the DNA was denatured at 94°C for 3 min were N80% homologous to their respective gene sequences from followed by 40
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