Isolation and characterization of eight microsatellites from the South American annual fish Austrolebias charrua
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MICROSATELLITE LETTERS
Isolation and characterization of eight microsatellites from the South American annual fish Austrolebias charrua Vero´nica Gutie´rrez • Silvana Pereyra Ne´stor Rı´os • Graciela Garcı´a
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Received: 13 June 2014 / Accepted: 7 July 2014 / Published online: 17 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Austrolebias charrua is an endemic annual fish of Uruguay and Brazil. Eight microsatellite markers were developed in order to clarify its population genetic structure. All of them constitute polymorphic loci. The number of observed alleles ranged from 6 to 14, the mean expected heterozygosity from 0.65 to 0.92 and the Shannon-Wiener Diversity Indices from 1.31 to 2.39. This study is the first report of microsatellites in annual killifish and they will be a helpful tool to implement conservation strategies in this endangered species. Keywords Austrolebias charrua Killifish South America Microsatellite loci Austrolebias charrua (Costa and Cheffe 2001) (Cyprinodontiformes: Rivulidae) is a South American annual killifish endemic of eastern Uruguay and Laguna dos Patos basin in Brazil. It lives in temporary ponds and each generation completes a full life cycle within one year. The eggs survive dry seasons buried in the mud and hatch in the subsequent rainy season (Wourms 1972). With a DNA content of 6.16 ± 0.28 (mean ± SE) and a cytotype of 2n = 48, this taxon exhibits extensive intraspecific karyotype divergences with different arm number (NF) values. Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0266-9) contains supplementary material, which is available to authorized users. V. Gutie´rrez (&) N. Rı´os G. Garcı´a Seccio´n Gene´tica Evolutiva, Facultad de Ciencias, Universidad de la Repu´blica, Montevideo, Uruguay e-mail: [email protected] S. Pereyra Departamento de Gene´tica, Facultad de Medicina, Universidad de la Repu´blica, Montevideo, Uruguay
According to DNA polymorphism estimates and chromosome data, this taxon shows the highest values of genetic variation and it could be considered as a hypothetic polymorphic ancestral taxon (Garcı´a et al. 2014). In order to clarify the population genetic structure to implement conservation strategies in this endangered species, new molecular markers were developed to complement existing mitochondrial gene analyses on this taxon. Genomic DNA was isolated from liver tissue of freshly sacrificed animals (fixed in ethanol 95 %) using a ‘‘salting out’’ protocol with sodium chloride protein precipitation and ethanol DNA precipitation. Enriched partial microsatellite genomic libraries for repeats motifs (GATA)10, (CA)13 and (GT)13 were obtained following the Glenn and Schable (2005) protocol with a few modifications. A total of 91 clones ([200 bp) were sequenced. All the sequences were analyzed in BatchPrimer3. Eleven of them contained 33 different microsatellite motifs. Compound microsatellites were discharged and eight primers pairs were created using Primer3 v. 0.4.0 softwar
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