The development and characterization of polymorphic microsatellite loci for the genus Melinaea (Nymphalidae, Ithomiini)
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MICROSATELLITE LETTERS
The development and characterization of polymorphic microsatellite loci for the genus Melinaea (Nymphalidae, Ithomiini) Melanie McClure • Mathieu Chouteau Bernard Angers • Marianne Elias
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Received: 10 May 2014 / Accepted: 19 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Due to their preference for undisturbed habitats, the butterflies of the genus Melinaea are promising indicators of ecological conditions. Here we describe 12 polymorphic microsatellite markers, with 3–26 alleles per locus, an observed heterozygosity of 0.138–0.889, and an expected heterozygosity of 0.400–0.970. These markers will prove useful in investigating patterns of differentiation in this clade. Keywords Aposematic Diversification Lepidoptera Mimicry Speciation The tribe Ithomiini (Nymphalidae: Danainae) represents a large group of Neotropical butterflies which numerically dominate forest communities. They prefer undisturbed forests, and have been proposed as promising biological indicators of ecological conditions and biological diversity. The species of the genus Melinaea are some of the largest ithomiines, but are difficult to separate from one another as a result of mimicry and polymorphism, and mitochondrial markers have failed to separate the different species
Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0233-5) contains supplementary material, which is available to authorized users. M. McClure (&) M. Chouteau M. Elias Institut de Syste´matique, E´volution et Biodiversite´, UMR 7205, Muse´um National d’Histoire Naturelle, CP50, 45 rue Buffon, 75005 Paris, France e-mail: [email protected] M. Chouteau B. Angers Department of Biological Sciences, Universite´ de Montre´al, C.P. 6128, Succ. CV, Montreal H3C 3J7, Canada
(Whinnett et al. 2005). Here we present primers that amplify 12 microsatellite loci for three closely related Melinaea species. Microsatellites were developed using 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries (Malausa et al. 2011) by GenoScreen (http://genoscreen.fr). Primers for candidate microsatellite loci were designed with the aid of Primer3 v.0.4.0 (Untergasser et al. 2012). DNA was extracted from thoracic tissue using Qiagen DNeasy tissue kits. PCR conditions (12.5 ll volume) were: 1 ll of 109 Taq Buffer, 0.2 mM dNTPs, 0.2 U Taq Polymerase (Qiagen), 5–40 ng DNA, 0.1 lM M13-tailed forward primer, 0.4 lM dye-labelled (NED, VIC, PET and 6-FAM) M13 primer (50 CACGACGTTGTAAAACGA C30 ), and 0.4 lM reverse primer. The PCR amplification temperature profile consisted of an initial denaturation at 92 °C for 2 min, 45 cycles at 92 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min, and a final elongation at 72 °C for 15 min (Table 1). PCR products were multiplexed (up to 4 loci) and 1 ll of the resulting solution was added to 8.8 ll Hi-Di formamide (Life Technologies) and 0.2 ll size standard (Life technologies Genescan 500LIZ). Products were resolved on an ABI 3130 Genetic Analyzer capillary DNA
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