Molecular Ecology Notes (2003) 3, 277 – 279 doi: 10.1046/j.1471-8286.2003.00425.x © 2003 Blackwell Publishing Ltd Blackwell Publishing Ltd. PRIMER NOTE Microsatellites for the Tasmanian devil (Sarcophilus laniarius) MENNA E. JONES,* DAVID PAETKAU,* ELI GEFFEN† and CRAIG MORITZ* *Department of Zoology and Entomology, University of Queensland, Queensland 4072, Australia, †Institute for Nature Conservation Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel Abstract The Tasmanian devil (Sarcophilus laniarius), a medium-sized predator/scavenger, is the largest member of the short-lived carnivorous marsupial Family Dasyuridae. Now restricted to Tasmania, populations are impacted by habitat clearance and anthropogenic mortality and genetic studies could be of value in informing levels of genetic diversity, mating system, dispersal and the effects of natural and anthropogenic landscape features on gene flow. Microsatellite markers were isolated from a partial, size-selected genomic library that was enriched for microsatellite sequences. Primer pairs were developed for 11 polymorphic dinucleotide microsatellite loci that conform with Hardy–Weinberg equilib- rium and reveal moderate genetic variability across the species range. Keywords: CA repeats, carnivorous marsupial, mating system, microsatellite, population genetics, Tasmanian devil Received 13 December 2002; revision accepted 24 January 2003 The largest carnivorous marsupial, the Tasmanian devil (Sarcophilus laniarius; Dasyuridae), is a medium-sized (males 8–13 kg; females 5–9 kg) predator and specialized scavenger in a family notable for short life spans (Cockburn 1997). Disappearing from the Australian mainland between 430 and 5000 years ago ( Jones et al. 2003), Tasmanian populations are reported to have reached very low densities for periods of a decade or more on at least three occasions in the last 150 years (Guiler 1992). Today, devils are displaced by over- clearing of native vegetation, are persecuted for livestock depredations and are vulnerable to dog attacks and road mortality ( Jones et al. 2003). Microsatellites will be used to examine the effects of historic periods of low population density on genetic variation, of natural and anthropo- genic habitat fragmentation on gene flow and population differentiation and to study dispersal, mating systems and life history through assignment tests and parentage analysis. Tasmanian devil genomic DNA was isolated using phenol–chloroform extraction (Sambrook et al. 1989) from ear biopsies stored at -20 °C in 70% ethanol. A size-selected partial genomic library was constructed using DNA from one male S. laniarius (Freycinet study animal no. 007) following the strand extension method of Paetkau (1999). Enrichment of tandem AC repeat microsatellites was achieved using an (AC) 18 5′-biotinylated oligonucleotide primer and produced 100% positive clones. Of 200 positive clones screened, 126 that showed strong visible poly- merase chain reaction (PCR) product on an agarose gel were sequenced using a Prism Ready Reaction Dye Cycle Sequencing kit and an ABI 373 automated sequencer (Applied Biosystems). Of these, 79 were rejected because they produced either messy or no sequence (n = 11), the microsatellite was short (52 with six to 14 repeats, six with two to five repeats), the microsatellite was complex or interrupted more than once (eight) or the flanking sequence was short (two). Forty-seven promising clones were reverse sequenced using a BigDye Terminator kit (ABI PRISM™)and an ABI 377. Seven duplicate clones were detected and rejected. Primers were designed for 23 loci which had long and near or actual continuous repeat sequences with the aid of oligo primer analysis soft- ware (National Biosciences, 1992). One of a primer pair was labelled with fluorescent dye (TET, FAM and HEX). A GTTT (or GT*) tail was added to the 5′ end of the unlabelled primer to promote complete adenylation (see Table 1). Correspondence: Menna E. Jones, School of Zoology, University of Tasmania, GPO Box 252-5, Hobart, Tasmania 7001, Australia. Fax: (613) 6226 2745; E-mail: menna.jones@utas.edu.au