Molecular Ecology Resources (2008) 8, 1451–1453 doi: 10.1111/j.1755-0998.2008.02200.x © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES Isolation and characterization of microsatellite markers for the ornamental discus fish Symphysodon discus and cross-species amplification in other Heroini cichlid species MANUELLA VILLAR AMADO,* TOMAS HRBEK,*† WALESKA GRAVENA,* CLEITON FANTIN,* ENEDINA NOGUEIRA DE ASSUNÇÃO,‡ SPARTACO ASTOLFI-FILHO‡ and IZENI P. FARIAS* *Departamento de Biologia, Laboratório de Evolução e Genética Animal, Universidade Federal do Amazonas, Av. Rodrigo Octávio Jordão Ramos, 3000, 69077-000, Manaus, Amazonas, Brazil, †Biology Department, University of Puerto Rico - Rio Piedras, San Juan, PR 00931, Puerto Rico, ‡Centro de Apoio Muldisciplinar, Laboratório de Tecnologias de DNA, Universidade Federal do Amazonas, Av. Rodrigo Octávio Jordão Ramos, 3000, 69077-000, Manaus, Amazonas, Brazil Abstract The discus fishes (Symphysodon spp.) are economically important ornamental species. Thirteen microsatellite markers were developed from a CT 12 - and CA 12 -enriched whole genomic DNA library of Symphysodon discus. Allelic variability was tested on 44 individuals of two species (S. discus and S. aequifasciatus). Allelic richness ranged from two to 11 alleles per locus and observed heterozygosities from 0.083 to 0.998. All loci were at Hardy–Weinberg equilibrium, and no pair of loci showed linkage disequilibrium within a species. Cross- species amplification was also successfully performed in the Neotropical cichlids Uaru amphiacanthoides, Hoplarchus psittacus, Hypselecara coryphaenoides, Pterophyllum sp., Mesonauta sp. and Heros sp. Keywords: Amazonia, Cichlidae, discus, microsatellites, ornamental fish, Symphysodon Received 4 November 2007; revision accepted 25 February 2008 The economically important fishes of the genus Symphysodon Heckel, 1840 originate from the Amazonian basin. They are grouped into five forms: ‘Heckel’ from the Negro and Trombetas River basins ´pineapple´ from the Abacaxis River, ‘green’ from the upper Amazon, ‘blue’ from the central Amazon and ‘brown’ from the lower Amazon. Based on meristic and colour-pattern data, ‘Heckel’ ‘pineapple’ correspond to Symphysodon discus Heckel, 1840 and remaining forms to Symphysodon aequifasciatus Pellegrin, 1904. Morphological and molecular analyses suggest a close phylogenetic relationship of Symphysodon with Uaru, Heros, Mesonauta and Pterophyllum (Farias et al. 2000); all species belong to the Heroini tribe of Neotropical cichlids (Kullander 2003). Since no microsatellite DNA markers are available for Symphysodon species or any other South American cichlids, we report the isolation and characterization of 13 micro- satellite markers for the discus fish. Loci were isolated using an enrichment technique (Farias et al. 2003). Briefly, genomic DNA from Symphysodon discus was extracted, completely digested with the restriction enzyme MboI (Fermentas), a 300–1000-bp size fraction was gel extracted, and fragments were ligated into an oligonucleotide adaptor and polymerase chain reaction (PCR) enriched using the inward extending adaptor primer. PCR products were denatured, and then hybridized to biotin-labelled CT 12 and CA 12 probes linked to DynaBeads (Invitrogen) via a biotin–streptavidin bond. Hybridized DNA–probe complex was washed in succes- sively more stringent standard saline citrate solutions, and finally the DNA was eluted off of the probe using a TE buffer. The enriched DNA was ligated into an Invitrogen pCR 2.1 TOPO cloning vector and transformed into com- petent Escherichia coli cells (TOPO TA Cloning kit). The transformed cells were grown overnight on 1× Luria- Bertani agar plates supplemented with 100 μg/mL of ampicillin (Sambrook & Russell 2001). Individual colonies were picked, and regrown in liquid Luria-Bertani/amp medium for 16 h in a 96-well culture plate. PCR amplification using M13 (–20) forward and M13 reverse primers was Correspondence: Izeni P. Farias, Fax: +55 (92) 3647-4229; E-mail: izeni_farias@ufam.edu.br