Molecular Ecology Notes (2006) 6, 859–861 doi: 10.1111/j.1471-8286.2006.01373.x © 2006 The Authors Journal Compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Isolation of polymorphic tetranucleotide microsatellite markers for the green-eyed tree frog (Litoria genimaculata) MELISSA M. GRAY,* CONRAD J. HOSKIN† and CAROLYNE BARDELEBEN* *Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 621 Charles Young Drive South, Los Angeles, California 90095-1606, USA, † School of Integrative Biology, University of Queensland, St Lucia, Queensland 4072, Australia Abstract We have isolated 16 polymorphic microsatellite markers for the green-eyed tree frog, Litoria genimaculata, from genomic libraries enriched for (AAGG) n and (AAAG) n repetitive elements. The number of alleles ranges from four to 14 per locus with the observed hetero- zygosity ranging from 0.36 to 1.00. These markers will be useful for analysis of questions concerning population genetic structure and speciation. Keywords: frog, Hylidae, Litoria, microsatellite, tetranucleotide Received 5 January 2006; revision received 27 January 2006; accepted 23 February 2006 The green-eyed tree frog, Litoria genimaculata, inhabits rainforest at all altitudes throughout the Wet Tropics of northeastern Queensland, Australia (Hero & Fickling 1994; Barker et al . 1995). As part of a collaborative project to understand how the diversity of species in rainforests has been generated, different models of speciation are being tested across multiple species (Moritz et al . 2000). As part of this effort, 16 polymorphic microsatellite markers for L. genimaculata have been developed. Microsatellite loci were isolated using a biotin-capture method, as previously described (Bardeleben 2004; Bardele- ben et al . 2004). Briefly, genomic DNA was isolated from liver and toe pad tissue using standard proteinase K diges- tion followed by extraction with phenol/chloroform and precipitated with ethanol (Sambrook et al . 1989). Approxi- mately 5 μ g of genomic DNA was restricted with Sau 3A or Bst YI, ligated to an adaptor generated by annealing together oligo A and oligo B (Refseth et al . 1997) and size fractionated on a 1% agarose/TAE gel. DNA in the 0.5 – 1.5 kilobase range was excised from the gel and the DNA purified by UltraClean (Mcfrugal). Approximately 5 μ g of genomic DNA was restricted with Mse I, ligated to an adaptor generated by annealing together oligo A and oligo C (Bardeleben 2004) and size fractionated as above. Lastly, approximately 5 μ g of genomic DNA was restricted with Msp I, ligated to an adaptor generated by annealing together oligo A and oligo D (5 ′ -PCGCGAAGCTTGGGGTCTCT- GGCC-3 ′ ) and size fractionated as above. Each hybridization was carried out with approximately 0.5 μ g of the adaptor- ligated, size-fractionated genomic DNA mixed with 50 n m biotin-labelled oligo probe [either 5 ′ -(AAGG) 6 GCA(Biotinyl- C)A-3 ′ or 5 ′ -(AAAG) 6 GCA(Biotinyl-C)A-3′]. Hybridizations were incubated in 6× SSC (1 m NaCl, 0.1 m NaCitrate) overnight. Hybridization was carried out at 65 °C with the AAGG probe and at 58 ° C for the AAAG probe. Subsequent steps including addition and incubation of 15- μ L aliquots of M280 streptavidin-coated magnetic beads (Dynal), washes, elution, a second round of hybridization and cloning were carried out exactly as previously described (Bardeleben 2004). A polymerase chain reaction (PCR)-based method previously described was used to screen the clones (Bardeleben et al . 2004). Plasmid DNA from 101 clones that were positive for a repetitive element were sequenced using ABI PRISM dGTP BigDye Ready mix (Applied Bio- systems) and either the M13( - 20) forward or M13altrp (Bardeleben et al . 2005) primer. Sequences were run on an automated DNA Sequencer (ABI PRISM 377, PE Applied Biosystems). Primers were designed from the flanking sequences of unique clones to amplify the repetitive element. PCR con- ditions were optimized for each primer set and each locus was evaluated for polymorphism and heterozygosity in a total of 14 individuals captured from various locations in northeastern Queensland, including Jum Rum Creek Con- servation Park (8 samples), Kowrowa (1), Streets Creek (2), Davies Creek (1), Palmerston (1) and Hinchinbrook Island Correspondence: Carolyne Bardeleben, Fax: 310-206-3987; E-mail: carolyne@ucla.edu