Development of polymorphic microsatellite markers for the Pleuroderma thaul
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Development of polymorphic microsatellite markers for the Pleuroderma thaul Guillermo D’Elı´a • Rochelle R. Beasley Stacey L. Lance • Kenneth L. Jones • Leonardo D. Bacigalupe
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Received: 13 March 2014 / Accepted: 10 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Pleurodema thaul (Leptodactylidae) is a small frog from Chile and Argentina with a distributional range that spans more than 2,500 km. We isolated and characterized a total of 23 microsatellite loci from 24 individuals. The number of alleles per locus ranged from 7 to 22, observed heterozygosity ranged from 0.417 to 0.958, and the probability of identity values ranged from 0.0064 to 0.21. Keywords Leptodactylidae Pleuroderma thaul Illumina Microsatellite PAL_FINDER PCR primers SSR Pleurodema thaul (Amphibia: Leiuperidae), a small endemic frog from Chile and Argentina (Vidal et al. 2009), is categorized as Least Concern in the IUCN redlist, although is considered to be vulnerable and endangered in the central and northern parts of its distributional range by the Chilean law. The species display a high degree of variation in phenotypic and life history traits and a shallow phylogeographic structure. Here, we report the development of 23 microsatellite loci for P. thaul, which can be used to assess the genetic diversity and population genetic G. D’Elı´a L. D. Bacigalupe (&) Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile e-mail: [email protected] R. R. Beasley S. L. Lance Savannah River Ecology Laboratory, University of Georgia, Aiken, SC 29802, USA K. L. Jones Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA
structure of this species throughout its distributional range for conservation and management purposes. Total DNA was extracted from one individual of P. thaul from Carrera Pinto, Chile (27°060 40.200 S, 69°530 44.300 W) for use in isolation of microsatellite loci. An Illumina paired-end shotgun library was prepared by shearing 1 lg of DNA using a Covaris S220 and following the standard protocol of the Illumina TruSeq DNA Library Kit and using a multiplex identifier adaptor index. Illumina sequencing was conducted on the HiSeq with 100 bp paired-end reads. Five million of the resulting reads were analyzed with the program PAL_FINDER_v0.02.03 (Castoe et al. 2012) to extract those reads that contained di-, tri-, tetra-, penta-, and hexanucleotide microsatellites. Once positive reads were identified in PAL_FINDER_v0.02.03 they were batched to a local installation of the program Primer3 (version 2.0.0) for primer design. To avoid issues with copy number of the primer sequence in the genome, loci for which the primer sequences only occurred one or two times in the 5 million reads were selected. Forty-eight loci of the 3,015 that met this criterion were chosen. One primer from each pair was modified on the 50 end with an engineered sequence (CAG tag
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