Molecular Ecology Notes (2004) 4, 321–323 doi: 10.1111/j.1471-8286.2004.00681.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE A multiplex panel of microsatellite markers for widespread sub-Saharan rodents of the genus Mastomys M. GALAN,* W. F. VAN HOOFT,† D. LEGRAND,* K. BERTHIER,* A. LOISEAU,* L. GRANJON* and J. F. COSSON* * Centre de Biologie et Gestion des Populations (UMR 22), INRA, Campus International de Baillarguet, CS 30016, 34988 Montferrier sur Lez cedex, France, † University of Antwerp, Department of Biology, Evolutionary Biology Group, Groenenborgerlaan 171, B-2020, Antwerpen, Belgium Abstract We isolated and characterized 12 polymorphic microsatellite loci in the sub-Saharan rodent Mastomys huberti. We tested cross-species amplification of all these loci in three closely related Mastomys species: M. coucha, M. erythroleucus and M. natalensis. Multiplex panels comprising 11 loci were developed and their application to a set of individuals in each species allowed clear and easy characterization of allele sizes. Statistics from 31 M. huberti coming from one locality in Mali showed no deviation from Hardy–Weinberg equilibrium except for one locus, and no significant linkage disequilibria between loci. Keywords: Mastomys coucha, M. huberti, M. erythrolocus, M. natalensis, microsatellites, multiplex- polymerase chain reaction, Rodentia Received 3 March 2004; revision accepted 25 March 2004 Multimammate rats of the genus Mastomys Thomas, 1915 are widely distributed throughout sub-Saharan Africa. They may display sporadic population explosions, making them important pests for standing crops and stored foods (Leirs 1994). Moreover, they are reservoirs and vectors of various human diseases (Gratz 1997) such as bubonic plague or Lassa fever. At least four Mastomys species are sibling species with no fully diagnostic external morphological criterion and karyotype remains the most reliable character to date for unambiguous species assignation (Granjon et al . 1997). Genetic studies (Duplantier et al . 1990a) and the finding of intermediate karyotypes in wild-caught Mastomys from Senegal (Duplantier et al . 1990b) suggest both ancient and contemporary hybridization events between some species. Although of great interest in the frame of pest control and human disease studies, gene flow rates within and between species are not known. Recently, we developed a molecular test based on polymorphism in the cytochrome b gene to assign individuals to one of the four sibling species (Lecompte et al . 2004). In the present study, we present data on the isolation of polymorphic microsatel- lites in M. huberti . Cross-species amplification tests were conducted on the three other Mastomys sibling species. Microsatellite screening was performed following Estoup & Cornuet (1994). Total DNA was extracted from muscles of a toe of one male M. huberti from Emnal’here (Mali) using silica columns with the Qiagen Tissue Kit. A partial genomic library was constructed by ligating 400 – 900 bp Sau 3 -AI-digested DNA into PUC-18 phagemic vector (Strata- gene) that was used to transform Epicurian Coli XL1-Blue supercompetent cells (Stratagene). Colonies were screened with two oligopolymers: (TC) 10 and (TG) 10 . We obtained 224 positive transformants over 2071 clones (10.8%). Among them, 96 were sequenced with ABI automated sequencer. We selected 20 sequences for which primers could be designed and used in multiplex reactions. We used a reaction volume of 10 μ L, containing about 50 ng of DNA (1 μ l DNA of one toe extraction), 0.2 μ m of each primer, 1.5 m m of MgCl 2 , 400 μm of dNTP and 1 U of Taq DNA Polymerase (Promega) and Thermophilic DNA Polymerase 10 × Buffer Mg Free (Promega). The polymer- ase chain reaction (PCR) started with a denaturation phase at 93 ° C for 2 min. We then performed 35 cycles with denaturation at 91 ° C for 30 s, annealing at 55 ° C for 30 s and extension at 74 ° C for 30 s (10 min for the last one). The PCR parameters were the same for each locus to facilitate subsequent amplification when multiplexing the primers. The amplification products were separated and detected by electrophoresis on large (15 × 25 cm) agarose Correspondence: Maxime Galan. Fax: + 33 4 99 62 33 45; E-mail: galan@ensam.inra.fr