A novel semi-multiplex PCR assay for identification of tropical eels of genus Anguilla in Indonesian waters
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ORIGINAL ARTICLE
Biology
A novel semi-multiplex PCR assay for identification of tropical eels of genus Anguilla in Indonesian waters Melta Rini Fahmi • Dedy Duryadi Solihin Kadarwan Soewardi • Laurent Pouyaud • Zhaojun Shao • Patrick Berrebi
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Received: 19 September 2012 / Accepted: 6 November 2012 / Published online: 3 January 2013 Ó The Japanese Society of Fisheries Science 2012
Abstract A one-step semi-multiplex polymerase chain reaction (PCR) is proposed to distinguish seven species and subspecies of tropical eels, i.e., Anguilla celebesensis, A. interioris, A. nebulosa nebulosa, A. marmorata, A. borneensis, A. bicolor bicolor, and A. bicolor pacifica, known in Indonesian waters. Seven pairs of species-specific primers, including two forward and seven reverse sequences, were designed after alignment of published and
M. R. Fahmi Animal Bioscience Study Program, Graduate School, Bogor Agricultural University, Bogor, Indonesia M. R. Fahmi (&) Researcher at Research Station of Ornamental Fish Culture, Jl Perikanan No 13 Pancoran Mas, Depok, Indonesia e-mail: [email protected] D. D. Solihin Department of Biology, Faculty of Mathematics and Natural Science, Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia e-mail: [email protected] K. Soewardi Department of Fisheries Resources, Faculty Marine and Fisheries, Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia e-mail: [email protected] L. Pouyaud Institut de Recherche pour le De´veloppement, UR 175, 361 rue J.F. Breton, 34196 Montpellier 5, France e-mail: [email protected] Z. Shao P. Berrebi Institut des Sciences de l’Evolution (ISEM) UMR 5554 UMII-CNRS-IRD, Universite´ Montpellier II, CC 065, Place E. Bataillon, 34095 Montpellier Cedex 5, France e-mail: [email protected]
new complete mitochondrial cytochrome b (1,140 bp) and 16S ribosomal RNA (rRNA) (1,120 bp) sequences. All species-specific primer pairs are included in one PCR mix, but only one pair of them can amplify a specific fragment from the template DNA that is analyzed. The semi-multiplex PCR amplified a fragment of 230 bp for A. b. bicolor, 372 bp for A. n. nebulosa, 450 bp for A. borneensis, 620 bp for A. marmorata, 670 bp for A. b. pacifica, 720 bp for A. celebesensis, and 795 bp for A. interioris, which are then separated by DNA agarose gel electrophoresis. Keywords Semi-multiplex PCR Anguilla spp. Tropical eels Molecular identification Species–subspecies-specific primer
Introduction The order Anguilliformes includes 141 genera and around 800 species, most of them living in the oceans [1]. The genus Anguilla is composed of freshwater eels having a catadromous life history, characterized by spawning in ocean waters and larval migration back to the parents’ growing habitats in estuarine or freshwater areas. The temperate species spawn in remote tropical waters after a long adult migration. Their larvae—called leptocephali— passively return to their growth habitat under the influence of subtropical currents, and therefore
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