PCR-RFLP and Sequencing of trnS/trnfM Fragment of Enhalus acoriodes from Sanur Coastal Waters, Bali, Indonesia: A Preliminary Study


  • Made Pharmawati Biology Department Faculty of Mathematics and Natural Sciences Udayana University Kampus Bukit Jimbaran, Bali
  • Eka Fibayani Imaniar Biology Department Faculty of Mathematics and Natural Sciences Udayana University Kampus Bukit Jimbaran, Bali




Enhalus acoroides, PCR-RFLP, Sequencing trnS/trnfM, Sanur


This study aimed to evaluate the resolution of PCR-RFLP and sequencing of trnS/trnfM in detecting genetic diversity of seagrass Enhalus acoroides at Sanur Beach, Sindhu Beach and Semawang Beach. This research used six samples E. acoroides from each location. DNA extraction method followed Doyle and Doyle. PCR amplifications were done using primers P672/P673, P674/P675 and ITS4/ITS5 followed by digestion using restriction enzymes EcoRI, PstI, HindIII, BamHI, Rsa, Mva and HinfI. Sequencing analysis used PCR products of primers trnS/trnfM. Testing of DNA sequences of E. acoroides were conducted using BLAST (Basic Local Alignment Search Tool). DNA sequences were further analyzed using MEGA 5.2 software (Molecular Evolutionary Genetic Analysis) to evaluate variations of DNA. The sequence alignments were done using ClustalW software to determine the homology between the DNA sequences. The results showed that 18 samples of E. acoroides from Sanur, Sindhu and Semawang Beach have no polymorphism based on restriction enzyme analyses. Furthermore, sequencing of trnS/trnfM region of 18 samples E. acoroides showed that the sequences were identical.   

Author Biographies

Made Pharmawati, Biology Department Faculty of Mathematics and Natural Sciences Udayana University Kampus Bukit Jimbaran, Bali

Jurusan BiologiFakultas Matematika dan Ilmu Pengetahuan AlamUniversitas UdayanaKampus Bukit Jimbaran, Bali

Eka Fibayani Imaniar, Biology Department Faculty of Mathematics and Natural Sciences Udayana University Kampus Bukit Jimbaran, Bali

Biology DepartmentFaculty of Mathematics and Natural SciencesUdayana UniversityKampus Bukit Jimbaran, Bali


Kiswara W, Winardi (1999) Sebaran lamun di Teluk Kuta dan Teluk Gerupuk, Lombok. Jakarta, Pusat Penelitian Oseanografi-LIPI.

Duarte CM, Sintes T, Marba N (2013) Assessing the CO2 capture potential of seagrass restoration projects. J App Ecol. doi:10.1111/1365-2664.12155.

Barbier EB, Hacker SD, Kennedy C et al (2011) The value of estuarine and coastal ecosystem services. Ecol Monograph 81(2): 169-193.

Andre J, Gyuris E, Lawder IR (2005) Comparison of the diets of sympatric dugongs and green turtles on the Orman Reefs, Torres Strait, Australia. Wildlife Res. 32:53-62.

Nienhuis PH, Coosen J, Kiswara W (1989) Community structure and biomass distribution of seagrasses and macrofauna in the Flores Sea, Indonesia. Neth J Sea Res. 23(2): 197-214.

Kuriandewa TE (2009) Tinjaun tentang Lamun di Indonesia. Lokakarya Nasional I Pengelolaan Ekosistem Lamun. Jakarta, Sheraton Media.

Yusup DS, Asy’ari H (2010) Komunitas tumbuhan lamun di kawasan perairan sekitar Denpasar. In Proceedings of the Seminar Nasional Biologi: Biodiversitas dan Bioteknologi Sumberdaya Akuatik. Purwokerto. Edited by Prabowo, Ardli ER, Sastranegara MH, Lestari W, Wijayanti G, Nuryanto A.

Nurkamila US (2014) Filogenetik lamun di Pantai Sanur dan Shindu berdasarkan penanda kloroplas DNA. Bachelor Thesis. Udayana University, Biology Department.

Kiswara W (2004) Kondisi padang lamun (seagrass) di Perairan Teluk Banten 1998-2001. Lembaga Penelitaian Oseanografi. Jakarta, Indonesian Institute of Science.

Arthana IW (2004) Spesies dan kerapatan padang lamun di Pantai Sanur Bali. Bumi Lestari J Env. 5(2): 1-11.

Outram MA, Jones EE, Jaspers MV et al (2014) Development of a PCR-RFLP method to distinguish species within the Ilyonectria macrodidyma complex. New Zealand Pl Protec. 67: 151-156.

Coutinho JP, Carvalho A, José Lima-Brito (2014) Taxonomic and ecological discrimination of Fagaceae species based on ITS PCR-RFLP. AoB PLANTS plu079 doi:10.1093/aobpla/plu079.

Sarin B, Martín JP, Kaula BC, et al (2015) Chloroplast DNA variations in wild Brassicas and their implication in breeding and population genetics studies. Scientifica, Vol 2015, Article ID 952395, doi: 10.1155/2015/952395.

Pharmawati M, Yan G, Sedgley R et al (2004) Chloroplast DNA inheritance and variation in Leucadendron species (Proteaceae) as revealed by PCR-RFLP. Theor Appl Genet. 109: 1694-1701.

Kress WJ, Wurdack KJ, Zimmer E et al (2005) Use of DNA barcodes to identify flowering plants. Proc Nat Acad of Sci USA. 102: 8369–8374.

Peay KG, Kennedy PG, Bruns TD (2008) Fungal community ecology: a hybrid beast with a molecular master. Bio. Sci. 58: 799–810.

Glick BR, Pasternak JJ, Patten CL (2010) Molecular biotechnology: Principles and applications of recombinant DNA 4th ed. Washington DC, ASM Press.

Kamiya K, Nanami S, Kenzo T et al (2012) Demographic history of Shorea curtisii (Dipterocarpaceae) inferred from chloroplast DNA sequence variations. Biotropica 44:577–585.

Sarra C, Soumaya RC, Zined M et al (2015) Chloroplast DNA analysis of Tunisian pistachio (Pistacia vera L.): Sequence variations of the intron trnL (UAA). Sci Hort. 191: 57-64.

Liu PL, Wan Q, Guo YP et al (2012) Phylogeny of the genus Chrysanthemum L.: Evidence from single-copy nuclear gene and chloroplast DNA sequences. PLoS ONE 7(11): e-48970. Doi:10.1371/journal.pone.0048970.

Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, United State America.

Lucas C, Tangaradjou T, Papenbrock J (2012) Development of a DNA barcoding system for seagrasses: succsesful but not simple. PloS ONE 7(1): e29987.

White TJ, Bruns T, Lee S et al (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. New York, Academic Press.

Demesure B, Sodzi N, Petit RJ (1995) A Set of universal primers for amplification of polymorphic non coding regions of mitochondrial and chloroplast DNA in plants. Mol Ecol. 4: 129–131.

Lowe A, Harris S, Ashton P (2006) Ecological Genetics (Design, Analysis and Application). Singapore, Blackwell publishing.

Gao H, Jiang K, Geng Y et al (2012) Development of microsatellite primers of the largest seagrass, Enhalus acoroides (Hydrocharitaceae). Amer J Bot. e99–e101.

Diaz-Almela E, Arnaud-Haond S, Vliet MS et al (2007). Feed-backs between genetic structure and perturbation-driven decline in seagrass (Posidonia oceanica) meadows. Cons Genet. 8:1377-1391.

Waycott M (2000) Matting system and population genetics of marine angiosperm. Wilson, K.L. and Morrison, DA. (eds). Monocots: Systematics and Evolution. Melbourne, CSIRO.

Lacap CDA, Vermaat JE, Rolton RN et al (2002) Propagule dispersal of the SE Asian seagrasses Enhalus acoroides and Thalassia hemprichii. Mar Ecol Prog Ser 235:75-80.

Capiomont A, Sandmeier M, Caye G et al (1996) Enzyme polymorphism in Posidonia oceanica, a seagrass endemic to the Mediterranean. Aqua Bot. 54: 265–277.

Procaccini G, Alberte RS, Mazzella L (1996) Genetic structure of the seagrass Posidonia oceanica in the western Mediterranean: ecological implications. Mar Ecol Prog Ser. 140: 153–160.

Arnaund-Haond S, Alberto F, Teixeira S et al (2005) Assessing genetic diversity in clonal organism: Low diversity or low resolution? Combining power and cost efficiency in selecting markers. J Hered. 96: 434-440.