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DNA Barcoding of Sangihe Nutmeg (Myristica Fragrans) Using Matk Gene

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DNA Barcoding of Sangihe Nutmeg (Myristica Fragrans) Using Matk Gene HAYATI Journal of Biosciences January 2015 Available online at: Vol. 22 No. 1, p 1-7 http://journal.ipb.ac.id/index.php/hayati ISSN: 1978-3019 DOI: 10.4308/hjb.22.1. DNA Barcoding of Sangihe Nutmeg (Myristica fragrans) using matK Gene TRINA EKAWATI TALLEI*, BEIVY JONATHAN KOLONDAM Department of Biology, Faculty of Mathematics and Natural Sciences, University of Sam Ratulangi, Kampus UNSRAT Bahu, Manado 95115, Indonesia Received September 3, 2014/Accepted December 22, 2014 Nutmeg (family: Myristicaceae) is a plant that originated from Banda islands and is widely cultivated in several places in the world. Secondary metabolites of this plant have a high value because of their benefits for the health, food, and beauty industries. This study aims at developing DNA barcode for nutmeg (Myristica fragrans) using standard recommended fragment of matK (maturase K) gene. Universal matK primer pairs were used to amplify 889 bp DNA fragment. BLAST search from NCBI site showed that Sangihe nutmeg has 100% identity with Myristica fatua, M. maingayi, and M. globosa. It also has 3 nucleotides difference with Rivola sebifera (identity 99.58%) and 4 nucleotides difference with Knema laurina (identity 99.43%). It can be inferred from this study that single locus of matK gene cannot be used to differentiate species in Myristica; it can only be used to differentiate the genus level within family Myristicaceae. Key words: Sangihe nutmeg, DNA barcode, matK, Myristica ___________________________________________________________________________ INTRODUCTION used (Schori & Showalter 2011). DNA barcode is a short sequence of a gene, usually less than 700 bp, Nutmeg family (Myristicaceae) comprises of which have been agreed upon and used to identify many species, which is widely spread in tropical rain species based on the references contained in the DNA forests around the world. This plant has ecological sequence database (Dick & Kress 2009). and ethnobotanical significances (Steeves 2011), and One of the challenges in the selection of this is amongst older group within angiospermae, which barcode is to distinguish very closely related or newly consists of recently evolved species (Newsmaster developed species. A species cannot be precisely & Ragupathy 2009). One member of this family is identified using DNA barcodes if the variation within Myristica fragrans (Houtt.), which was originated barcode between species is low, or related species still from eastern part of Indonesia (North Sulawesi and retain ancestral polymorphism, or they have a history Mollucas). Identification of species from nutmeg of hybridization (Dick & Kress 2009). Ideally, a DNA family is difficult considering the similarity of barcode should be routinely used using a primer pair, their leaves, so identification relies heavily on which can also be used for bidirectional sequencing, the characteristics of small flowers (1-4 mm in and provides the maximum level of discrimination size), which can only be obtained when the plant between species (Janzen 2009). is mature sufficiently (Newsmaster & Ragupathy Many genes used in plant systematics are in 2009). Identification of nutmeg is needed to mitigate substitution rate spectrum, representing genes that contamination, substitution, and falcification of evolve rapidly or slowly. Which genes to be used, herbal products, as WHO suggested that falcification is usually determined by the level of phylogenetic of herbal products is a threat to consumer safety analysis conducted by researcher. Each region in a (Newsmaster et al. 2013). gene has strengths and weaknesses. Good quality A previous research about Myristicaceae showed sequences, for example, can be found in the rbcL that misidentification of spesies in this family was (ribulose-1, 5-bisphosphate carboxylase oxygenase more than 50% in herbarium specimen and 25% in large subunit) and atpB (ATPase beta-subunit), but ecological plot (Steeves 2011). Due to the difficulty these sequences have a low level of differentiation in identifying the morphology, especially for cryptic of species because they are highly conserved among species, DNA diagnostic tool including plastid DNA plant groups, therefore its resolution is only good in _________________barcode and multilocus genomic marker can be the level of family and above. Sequences that have ∗Corresponding author. Phone: +62-431-827932, high degree of species differentiation are trnH- Fax: +62-431-853715, E-mail: [email protected] psbA (chloroplast intergenic spacer) and matK 2 TALLEI AND KOLONDAM HAYATI J Biosci (maturase K), because it evolves so quickly that DNA Amplification. Primers used for DNA provides enough character to analyze evolution below amplification of matK gene were MatK-1RKIM-f family level (Barthet 2006; Hollingsworth 2011). 5’-ACCCAGTC CATCTGGAAATCTTGGTTC-3’ The matK gene is considered to evolve rapidly, and MatK-3FKIM-r 5’-CGTACAGTACTTTTGT due to the fact that the gene has a high degree of GTTTACGAG-3’ designed by K. J. Kim from substitution and its sequence is more varied than School of Life Sciences and Biotechnology, Korea other genes (Barthet 2006). However, a group of University, Seoul, Korea (Kuzmina et al. 2012). researcher at the Consortium for the Barcode of Life DNA was amplified using 5x Firepol PCR Master (CboL) recommends two loci combination, rbcL and Mix Ready-to-Load (Solis BioDyne). Total volume matK, as standard DNA barcode for plants. These two for amplification was 40m L consisted of 2 mL DNA regions in chloroplast DNA were chosen because of sample and 1.5 mL of each primer (10 mM). DNA having high degree of differentiation between species amplification was carried out in PCR TPersonal (Bafeel et al. 2011). Furthermore, these two genes (Bimetra) as follows: predenaturation at 95oC for 4 play important role in phylogenetic reconstruction min followed by 35 cycles of denaturation at 95oC for for land plants (Kuzmina et al. 2012). 30 sec, annealing at 50 oC for 40 seconds, elongation Species identification in Myristicaceae family is at 72 oC for 50 sec, and one cycle of final extention important in order to distinguish invasive species at 72 oC for 2 min. The PCR products were separated from endangered species, or species that has an by 1.0% agarose gel electrophoresis. Amplicon was economic significance. This study aims at analyzing bidirectionally sequenced at First BASE (Malaysia). matK gene as a candidate for DNA barcode in nutmeg Data Analysis. Sequence chromatogram was family. edited using Geneious 6.1.6 (Biomatters Ltd., Auckland, New Zealand) and assembled into MATERIALS AND METHODS bidirectional contig. Primer sequences were removed, reverse and complement process were conducted DNA Extraction. Fresh leaf of Sangihe nutmeg using primer MatK-3FKIM-r, and combined using was extracted using innuPrep DNA Micro Kit MUSCLE (Multiple Sequence Comparison by Log- (Analytik Jena) according to the protocol given by Expectation), which is integrated within Geneious. the company. Five mg of fresh leaf was cut into The matK gene sequence was employed in small pieces and placed in a 1.5 mL Eppendorf tube. Basic Local Alignment Search Tool (BLAST) Into the tube, 200 mL Lysis Solution TLS and 20 mL (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to query Proteinase K were added. The tube was vortexed for highly similar sequence. Correct identification vigorously for 5 seconds and incubated at 50 oC for means identity percentage of BLAST is the highest 30 min. Tube was centrifuged at 12,000 rpm for 1 searched sequences derived from expected species, or min and supernatant was transferred into new 1.5 mL species from expected genus. In contrary, ambigous tube. Into the supernatant, 200 mL Binding Solution identification means identity percentage of BLAST TBS was added to lyse the sample then vortexed. is the highest of the searched sequenced derived not Sample was applied to Spin Filter located in a 2.0 from expected species or genus, or family. A final mL Receiver Tube and centrifuged at 12,000 rpm for alignment for matK sequences was generated using 1 min. Into the Spin Filter, 400 mL Washing Solution Multalin V.5.4.1 developed by Corpet (1988) (http:// was added and centrifuged at 12,000 rpm for 30 multalin.toulouse.inra.fr/ multalin/). Sequences were sec. Receiver Tube with filtrate in it was discarded trimmed at both ends of the alignment to avoid many and Spin Filter was placed in a new Receiver Tube. missing data at the end of the sequences so the final Into the Spin Filter, 750 mL Washing Solution MS characters were 707 sequences. Phylogenetic tree was was added and the tube was centrifuged at 12,000 constructed using software developed by Dereeper rpm for 30 sec. The Receiver Tube containing filter et al. (2008) (http://www.phylogeny.fr) and NCBI was discarded and Spin Filter was again placed in (http://ncbi.nlm.nih.gov). Similarity percentage a new Receiver Tube. The tube was centrifuged for matrix was calculated based on Clustal 2.1 (www. maximum speed for 2 min to remove all traces of ebi.ac.uk) ethanol. The receiver tube was discarded. The Spin Filter was added to Elution Tube and 100 mL of RESULTS Elution Buffer was added. The tube was incubated for 1 min at room temperature and centrifuged at 8,000 We successfully amplified partial matK gene rpm for 1 min. The eluted DNA was used for DNA from chloroplast genome of Sangihe nutmeg for the amplification. development of nutmeg DNA barcode (Figure 1). Vol. 22, 2015 DNA Barcoding of Sangihe Nutmeg 3 Its sequence was used as query sequence in BLAST M. globosa (100%), Virola sebifera (99.58%), and at NCBI to find similar sequence under the same or Knema laurina (M. laurina) (99.43%) (Table 1). This different genus within family Myristicaceae. The finding confirms that Sangihe nutmeg isM. fragrans. most highly similar identity sequences obtained from It has similarity 99.58% with Kneuma laurina the GenBank are M. fragrans, M. maingayi, M. fatua, (synonym of M.
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