MICROSATELLITE LETTERS Development of microsatellites for Southern Darwin’s frog Rhinoderma darwinii (Dume ´ril & Bibron, 1841) Marina Fuentes-Navarrete • Daniel Gomez-Uchida • Cristian Gallardo-Escarate • Cristian B. Canales-Aguirre • Ricardo Galleguillos • Juan Carlos Ortiz Received: 20 June 2014 / Accepted: 7 July 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract The Southern Darwin’s frog Rhinoderma darwinii is a charismatic, mouth-brooding amphibian endemic to temperate forests of South America with a Vulnerable conservation status according to the IUCN Red List. We developed microsatellite markers from next gen- eration sequence data that will aid genetic monitoring during and after re-introduction efforts. Using bioinfor- matics we characterized 3,521 perfect microsatellite repeats and designed primers for 35 of them. From these, 23 were polymorphic and amplified reliably. Number of alleles varied between 2 and 15, allele sizes varied between 84 and 299 bp, and observed heterozygosities varied between 0.105 and 0.904. These microsatellites represent a valuable resource to aid recovery of threatened Southern Darwin’s frog populations. Keywords Southern Darwin’s frog  Rhinoderma darwinii  Microsatellites  Next generation sequencing Populations of Southern Darwin’s frog Rhinoderma dar- winii (Amphibia: Rhinodermatidae), a charismatic, mouth- brooding amphibian endemic to temperate forests of South America, have decreased dramatically in recent decades, likely due to changes in land use, habitat fragmentation and chytridiomycosis (Ortiz and Heatwole 2010; Soto-Azat et al. 2013). Their current conservation status is set to Vulnerable according to the IUCN Red List (http://www. iucnredlist.org). Conservation measures and ex situ captive breeding initiatives are underway to aid re-introduction efforts, which can be assisted by the monitoring of genetic diversity. Here we report 23 polymorphic microsatellite DNA loci to implement genetic monitoring for R. darwinii. Total genomic DNA was extracted from muscle tissue taken from a single individual (RdaCB10) preserved at Estacio ´n de Monitoreo y Reproduccio ´n Rana de Darwin (EMRRD), Universidad de Concepcio ´n. We used a salting- out protocol followed by phenol–chloroform purification and ethanol precipitation steps. Shotgun sequencing on a GS Junior System (Roche) was outsourced to OMICS SOLUTIONS (www.omics-solutions.cl) and generated 118,506 reads with an average length of 399 bp. QDD v 2.1 (Megle ´cz et al. 2010) was used to find 3,521 perfect microsatellites (mostly di-, tri-, and tetra-nucleotide repeats) containing suitable flanking regions and located outside regions with high prevalence of mobile and trans- posable elements. Optimal settings for primer design included: (1) 20–21 nucleotides in length, (2) similar G?C content (47–50 %) between each primer of the pair, and (3) melting temperature of 59 °C. We calculated Gibbs free energy in 150 randomly chosen primer pairs using the M. Fuentes-Navarrete  J. C. Ortiz (&) Laboratorio de Herpetologı ´a, Biodiversidad y Ecologı ´a Molecular, Departamento de Zoologı ´a, Facultad de Ciencias Naturales y Oceanogra ´ficas, Universidad de Concepcio ´n, Casilla 160-C, Concepcio ´n, Chile e-mail: jortiz@udec.cl D. Gomez-Uchida Genomics in Ecology, Evolution and Conservation Laboratory, INCAR, Departamento de Zoologı ´a, Facultad de Ciencias Naturales y Oceanogra ´ficas, Universidad de Concepcio ´n, Casilla 160-C, Concepcio ´n, Chile C. Gallardo-Escarate Laboratory of Biotechnology and Aquatic Genomics (INCAR), University of Concepcio ´n, P.O. Box 160-C, Concepcio ´n, Chile C. B. Canales-Aguirre  R. Galleguillos Laboratorio de Gene ´tica y Acuicultura, Departamento de Oceanografı ´a, Facultad de Ciencias Naturales y Oceanogra ´ficas, Universidad de Concepcio ´n, Casilla 160-C, Concepcio ´n, Chile 123 Conservation Genet Resour DOI 10.1007/s12686-014-0279-4 Author's personal copy