Molecular Ecology Notes (2002) 2, 498 – 500 doi: 10.1046/j.1471-8278 .2002.00290.x © 2002 Blackwell Science Ltd Blackwell Science, Ltd PRIMER NOTE Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles funestus A. COHUET,* F. SIMARD,† A. BERTHOMIEU,‡ M. RAYMOND,‡ D. FONTENILLE* and M. WEILL‡ *Laboratoire de Lutte contre les Insectes Nuisibles (LIN), Institut de Recherche pour le Développement (IRD), BP 64501, 34394 Montpellier Cedex 05, France, †Laboratoire IRD d’Entomologie Médicale, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), BP 288, Yaoundé, Cameroun, ‡Institut des Sciences de l’Evolution, Laboratoire de Génétique et Environnement, CC065, UMR CNRS 5554, Université de Montpellier II, France Abstract Screening of the Anopheles funestus genomic DNA library detected 18 new sequences with dinucleotide tandem repeats. Primers were designed to amplify the loci and 14 out of 18 gave a repeatable and scorable amplification. Deviations from Hardy–Weinberg expectations were tested for each locus in a sample of 30 wild Anopheles funestus females. No hetero- zygote deficiency was detected for 11 loci of 14, thus revealing the absence of null alleles. The number of alleles per locus ranged from 5 to 15, and observed heterozygosity from 0.13 to 0.85. Keywords: Anopheles funestus, malaria, microsatellites, polymorphism Received 18 June 2002; revision received 12 July 2002; accepted 12 July 2002 Anopheles funestus is an important vector of human malaria in sub-Saharan Africa, being the main vector in some areas. Chromosomal inversion polymorphism suggests a genetic substructure within wild A. funestus populations, sometimes at a microgeographic scale (Costantini et al. 1999; Dia et al. 2000). Neutral molecular markers are needed to decipher further the genetic population structure. Twenty-two microsatellite DNA loci were recently characterized by Sinkins et al. (2000) but additional loci would be required for high throughput investigation of the distribution of neutral variability, within and between natural A. funestus populations. Development of new markers would eventually benefit gene mapping and ‘quantitative trait loci’ analysis in this important malaria vector. This study reports the characterization of a new set of polymorphic microsatellite markers. Microsatellite loci were isolated as described by Estoup et al. (1993) using the detailed protocols of A. Estoup and O. Martin that are available at HTTP://www.inapg. inra.fr/dsa/microsat/microsat.htm. Genomic DNA was extracted from a pool of 20 A. funestus specimens and was totally digested by Sau3A. Size-selected fragments (400 – 900 base pairs) were ligated into a pUC18 vector (Pharmacia) digested by BamHI, and plasmids were used to transform XL1-blue competent cells (Stratagene). Approx- imately 3000 recombinant clones were transfered onto Hybond-N + nylon membranes (Amersham) and screened with an equal mixture of (TC) 10 and (TG) 10 digoxigenine end-labelled oligonucleotide probes (Boehringer Mannheim). Plasmid DNA from 21 positive clones was purified using QIAprep Spin Miniprep Kit™ (Qiagen) and sequences of inserts were obtained with an ABI 310 sequencer (Perkin-Elmer). Among 21 sequences, three were identical to formerly described loci (Sinkins et al. 2000). Polymerase chain reaction (PCR) primers were designed flanking each of 18 remain- ing microsatellite sequences using the computer program oligo™ (version 4.0, National Biosciences). Primer pairs were chosen to amplify short (80–240 base pairs) PCR products. Microsatellite variability was analysed using 30 females from Cameroon. DNA was isolated from single specimens following Collins et al. (1987). PCR amplifications were carried out in a 25-μL reaction volume, from approximately 5–10 ng of template DNA. The reaction mixture contained 1× Qiagen PCR buffer (1.5 mm MgCl 2 ), 200 μm each dNTP, 10 pmol of each primer, and 1 U Qiagen Taq Polymerase. The forward primer was 5′ modified with either TET, HEX, or FAM fluorescent labels (Eurogentec) to allow multiplex Correspondence: Anna Cohuet. Fax: + 33 4 67 54 20 44; E-mail: anna.cohuet@mpl.ird.fr