Molecular Ecology Resources (2009) 9, 350–353 doi: 10.1111/j.1755-0998.2008.02389.x © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd Blackwell Publishing Ltd PERMANENT GENETIC RESOURCES NOTE Development of microsatellite markers in the Australasian snake-necked turtle Chelodina rugusa and cross-species amplification E. A. ALACS*, M. J. HILLYER†, A. GEORGES*, N. N. FITZSIMMONS§ and J. M. HUGHES† *Institute for Applied Ecology and National Centre for Forensic Studies, University of Canberra, Canberra ACT 2601, Australia, †Australian Rivers Institute, 170 Kessels Road, Nathan, Qld 4111, Australia, §Institute for Applied Ecology, University of Canberra, Canberra ACT 2601, Australia Abstract Seventeen microsatellite loci were developed for the snake-necked turtle, Chelodina rugosa (Ogilby, 1890). Sixteen of the loci were polymorphic but three of these loci had null alleles. One locus displayed linkage disequilibrium. These 17 markers were tested for amplification in eight congeneric species with varying success; 98% amplification in Chelodina burrun- gandjii, 72% in C. canni, 38% in C. expansa, 58% in C. longicollis, 67% in C. mccordi, 73% in C. oblonga, 81% in C. parkeri, and 68% in C. pritchardi. These microsatellite markers will be useful for population assignment, gene flow, mating systems and hybridization studies in the genus Chelodina. Keyword: Australia, Chelidae, Chelonia, freshwater turtle, hybridization, wildlife management Received 7 May 2008; revision accepted 4 June 2008 The genus Chelodina (Testudines: Chelidae) is an Australasian genus of snake-necked turtles comprising 13 species from Australia, Papua New Guinea, East Timor, Indonesian West Papua and Roti (Georges & Thomson 2006). Chelodina rugosa is found in parts of northern Australia, West Papua and southern Papua New Guinea. It is subject to legal harvest for the pet trade by the indigenous community of Maningrida in Arnhem Land, Northern Territory, Australia. We developed 17 microsatellite loci to test whether we could distinguish between legal collections of C. rugosa in Arnhem Land and illegal poaching activities. These loci were characterized for 76 individuals from two populations of C. rugosa from the Northern Territory that are 1.2 km apart (sample sizes of 41 and 35, respectively). We also tested the primers on eight other species: Chelodina burrungandjii, C. canni, C. expansa, C. longicollis, C. mccordi, C. oblonga, C. parkeri and C. pritchardi to better understand the complex patterns of hybridization that occur in this genus (Georges et al. 2002). Total genomic DNA was extracted from skin tissue samples (taken from vestigial hind toe webbing) using standard salting-out protocol (Dethmers et al. 2006). A genomic library enriched for di- and trinucleotide repeats was constructed based on the fast isolation by amplified fragment length polymorphism (AFLP) of sequences containing repeats (FIASCO) method (Zane et al. 2002). Modifications on the prescribed method are described below. DNA from a composite sample of four individuals (approximately 100 ng) was simultaneously digested with MseI and ligated to MseI AFLP adaptor (5′-TACTCAG- GACTCAT-3′/5′-GACGATGAGTCCTGAG-3′). The sub- sequent digestion–ligation mixture was amplified using polymerase chain reaction (PCR) under standard cycling conditions with the primer MseI-N (5′-GATGAGTCCT- GAGTAAN-3′). Amplified DNA was hybridized with a ‘pool’ of biotinylated probes [(AAC) 8 (ACC) 8 (AGC) 8 , & (ACG) 8 ] by mixing preheated hybridization buffer (181 μL 6× SSC, 3 μL 10% SDS, 6 μL 50× Denhards) with a de- natured solution containing 100 μL of amplified DNA and 10 μL of the probe pool. The total solution was incubated at 62 °C for 30 min. Hybridized DNA molecules were selec- tively captured using Streptavidin MagneSphere Paramagnet Particles (S-PMP) (Promega). Two hundred microlitres 6× SSC, 4 μL 50× Denhards and 2 μL 10% SDS, were added to the S-PMP, followed by the prepared DNA-probe hybridization and rotated for 20 min. The resultant S-PMP- probe-DNA conglomerate was then isolated using magnetic field separation. Removal of nonspecific DNA occurred through a sequence of two nonstringency washes followed Correspondence: Erika Alacs, Institute for Applied Ecology, University of Canberra, Canberra, ACT 2601, Australia. Email alacs@aerg.canberra.edu.au