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Myristica Fragrans) Using Matk Gene View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Scientific Journals of Bogor Agricultural University HAYATI Journal of Biosciences January 2015 Available online at: Vol. 22 No. 1, p 41-47 http://journal.ipb.ac.id/index.php/hayati EISSN: 2086-4094 DOI: 10.4308/hjb.22.1.41 SHORT COMMUNICATION 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 in identifying the morphology, especially for cryptic species, DNA diagnostic tool including plastid DNA Nutmeg family (Myristicaceae) comprises of barcode and multilocus genomic marker can be many species, which is widely spread in tropical rain used (Schori & Showalter 2011). DNA barcode is a forests around the world. This plant has ecological short sequence of a gene, usually less than 700 bp, and ethnobotanical significances (Steeves 2011), and which have been agreed upon and used to identify is amongst older group within angiospermae, which species based on the references contained in the DNA consists of recently evolved species (Newsmaster sequence database (Dick & Kress 2009). & Ragupathy 2009). One member of this family is One of the challenges in the selection of this Myristica fragrans (Houtt.), which was originated barcode is to distinguish very closely related or newly from eastern part of Indonesia (North Sulawesi and developed species. A species cannot be precisely Mollucas). Identification of species from nutmeg identified using DNA barcodes if the variation within family is difficult considering the similarity of barcode between species is low, or related species still their leaves, so identification relies heavily on retain ancestral polymorphism, or they have a history the characteristics of small flowers (1-4 mm in of hybridization (Dick & Kress 2009). Ideally, a DNA size), which can only be obtained when the plant barcode should be routinely used using a primer pair, is mature sufficiently (Newsmaster & Ragupathy which can also be used for bidirectional sequencing, 2009). Identification of nutmeg is needed to mitigate and provides the maximum level of discrimination contamination, substitution, and falcification of between species (Janzen 2009). herbal products, as WHO suggested that falcification Many genes used in plant systematics are in of herbal products is a threat to consumer safety substitution rate spectrum, representing genes that (Newsmaster et al. 2013). evolve rapidly or slowly. Which genes to be used, A previous research about Myristicaceae showed is usually determined by the level of phylogenetic that misidentification of spesies in this family was analysis conducted by researcher. Each region in a more than 50% in herbarium specimen and 25% in gene has strengths and weaknesses. Good quality ecological plot (Steeves 2011). Due to the difficulty _________________ sequences, for example, can be found in the rbcL ∗Corresponding author. Phone: +62-431-827932, (ribulose-1, 5-bisphosphate carboxylase oxygenase Fax: +62-431-853715, E-mail: [email protected] large subunit) and atpB (ATPase beta-subunit), but 42 TALLEI AND KOLONDAM HAYATI J Biosci these sequences have a low level of differentiation of ethanol. The Receiver Tube was discarded. The of species because they are highly conserved among Spin Filter was added to Elution Tube and 100 mL of plant groups, therefore its resolution is only good in Elution Buffer was added. The tube was incubated for the level of family and above. Sequences that have 1 min at room temperature and centrifuged at 8,000 high degree of species differentiation are trnH- rpm for 1 min. The eluted DNA was used for DNA psbA (chloroplast intergenic spacer) and matK amplification. (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 land plants (Kuzmina et al. 2012). for 30 sec, annealing at 50 oC for 40 sec, 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 matrix was calculated based on Clustal 2.1 (www. for maximum speed for 2 min to remove all traces ebi.ac.uk) Vol. 22, 2015 Short Communication ������� 43 RESULTS Its sequence was used as query sequence in BLAST at NCBI to find similar sequence under the same or We successfully amplified partial matK gene different genus within family Myristicaceae. The from chloroplast genome of Sangihe nutmeg for the most highly similar identity sequences obtained from development of nutmeg DNA barcode (Figure 1). the GenBank are M. fragrans, M. maingayi, M. fatua, M. globosa (100%), Virola sebifera (99.58%), and + MF 1 kb Knema laurina (M.
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    1762 TROPICAL FORESTS / Myristicaceae Burtt Davy J (1938) Classification of Tropical Woody importance. Fruit of the Myristicaceae, particularly Vegetation Types. Oxford, UK: Imperial Forestry In- the lipid-rich aril surrounding the seed in some stitute. species, are important as food for birds and mam- Champion HG and Seth SK (1968) Revised Survey of the mals of tropical forests. Numerous species are valued Forest Types of India. New Delhi, India: Manager of by humans as sources of food, medicine, narcotics, Publications, Government of India. and timber, including Myristica fragrans, the source Collins NM, Sagyer JA, and Whitmore TC (eds) (1991) of nutmeg and mace, the spices of commerce. The Conservation Atlas of Tropical Forests: Asia and Pacific. London: Macmillan. Throughout the geographical range of Myristica- Edward MV (1950) Burma Forest Types. Indian Forest ceae, aromatic leaves, often stellate pubescence, a Records (new series). Silviculture 7(2). Dehra Dun, India: unique arborescent architecture (Figure 1), and sap Forest Research Institute. the color of blood (Figure 2) are characteristics that Holmer CH (1958) The broad pattern of climate and strongly enhance recognition of this family in the vegetation distribution in Ceylon. In Proceedings of a field. Species of this family are usually dioecious. Symposium on Humid Tropics Vegetation, Kanly. Paris: Flowers are tiny and found in paniculate inflores- UNESCO. cences, with filaments of stamens fused into a IUCN (1988) The Red Data Book. Switzerland: IUCN. column, giving rise to either free or fused anthers Kurz S (1877) Forest Flora of British Burma, 2 vols. (Figures 3 and 4). Fruits are one-seeded, dehiscent or Calcutta, India: Bishen Singh, Dehra Dun.
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