Molecular Ecology Notes (2002) 2, 443 – 445 doi: 10.1046/j.1471-8278 .2002.00272.x © 2002 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTE Characterization of tetranucleotide microsatellite loci in a Lake Victorian, haplochromine cichlid fish: a Pundamilia pundamilia × Pundamilia nyererei hybrid *MARTIN I. TAYLOR,* FIONA MEARDON,* GEORGE TURNER,† OLE SEEHAUSEN,†‡ HILARY D. J. MROSSO‡§ and CIRO RICO* *School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, Norfolk, UK, †Department of Biological Sciences, University of Hull, HU6 7RX, UK, ‡Institute for Evolutionary and Ecological Sciences, University of Leiden, PO Box 9516, 2300 RA Leiden, the Netherlands, §Tanzania Fisheries Research Institute (TAFIRI), PO Box 475 Mwanza, Tanzania Abstract The haplochromine cichlid fishes inhabiting Lake Victoria in East Africa are of great inter- est to evolutionary biologists. We have isolated and optimized six tetranucleotide and a sin- gle dinucleotide locus from a hybrid of the haplochromine cichlid fishes, Pundamilia pundamilia and P. nyererei. Characterization in 18 individuals of P. nyererei from a single wild population revealed between six and 18 alleles per locus, with expected heterozygos- ity ranging from 0.68 to 0.94. These loci will prove useful for investigations of population structure, and elucidating relationships between closely related species. An additional 26 unoptimized loci have been deposited with GenBank and the MEN database. Keywords: cichlid, haplochromine, microsatellites, Pundamilia Received 1 April 2002; revision received 9 May 2002; accepted 30 May 2002 The cichlid fishes inhabiting Lake Victoria represent one of the most spectacular vertebrate radiations on the planet. As many as 500 species are thought to have evolved within the last 200 000 years (Meyer 1993) or possibly as little as 14 600 years (Owen et al. 1990; Johnson et al. 1996, 2000), although see Fryer (2001) and Seehausen (2002). Environ- mental deterioration and the introduction of the Nile perch (Lates niloticus) led to a catastrophic extinction of many of the endemic cichlid species, although the impact on the rock-dwelling species appears to have been less devastat- ing (Seehausen et al. 1997). While numerous dinucleotide loci are available for use in African haplochromine cichlid species (van Oppen et al. 1997; Wu 1998) the majority are dinucleotide repeats, and none have been developed specifically for Lake Victorian haplochromine species. We present a suite of easily scored tetranucleotide loci for the rock-dwelling, haplochromine cichlids Pundamilia pundamilia and P. nyererei, both endemic to Lake Victoria. These loci will prove useful for both evolutionary research and conservation programmes focusing on the haplochro- mine cichlids of Lake Victoria. Whole genomic DNA was extracted from a single F1 aquarium hybrid specimen of P. pundamilia × Pundamilia nyererei using a salt extraction protocol modified from (Aljanabi & Martinez 1997). Total DNA was cut simultane- ously with RsaI restriction enzyme and ligated onto SNX oligonucleotide linkers (Hamilton et al. 1999) by incubating for 2 h at 37 °C. The mixture was amplified using poly- merase chain reaction (PCR), and the product hybridized with biotin-labelled (GATA) 10 oligonucleotides. The mixture was then enriched using streptavidin MagneSphere par- ticles (Promega, Southampton, UK), with three washes in Tris-buffered saline Tween-20 (TBST) (25 mm Tris/Tris·HCl, 0.15 m NaCl, pH 7.5, 0.05% (w/v) Tween-20), three washes in 0.2 × Sodium chloride/sodium citrate buffer (SSC) (30 mm NaCl, 3 mm sodium citrate, pH 7.0), the enriched DNA was eluted in 100 μL Tris low EDTA buffer (TLE) (10 mm Tris; 0.1 mm EDTA, pH 8.0). The enriched single- stranded DNA was immediately PCR recovered, PEG (poly- ethylene glycol) precipitated, and A-tailed by incubating 21.6 μL of PCR product at 72 °C for 30 min with 0.5 μL 10× PCR buffer (Bioline, London, UK), 0.4 μL MgCl 2 (25 mm), 1 μL dATP (10 mm), 0.2 μL Taq (Bioline), 2.2 μL H 2 O. The A-tailed PCR product was then ligated into a pGEM-T Easy Correspondence: Martin I Taylor. E-mail: nitram8@hotmail.com