Development of 31 new microsatellite loci for two mole salamanders ( Ambystoma laterale and A. jeffersonianum )
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MICROSATELLITE LETTERS
Development of 31 new microsatellite loci for two mole salamanders (Ambystoma laterale and A. jeffersonianum) Robert D. Denton • H. Lisle Gibbs Travis C. Glenn
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Received: 3 June 2014 / Accepted: 23 August 2014 / Published online: 29 August 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Ambystoma salamanders are amphibians that due to limited dispersal abilities and reliance on wetlands for breeding are susceptible to population declines and local extinctions (Blaustein et al. 2011). Species identification within Ambystoma is especially difficult due to the presence of unisexual Ambystoma that consist of multiple all-female lineages in which clones can have between two and five nuclear genomes from up to five other Ambystoma species (Bogart et al. 2007). The majority of these unisexual Ambystoma are composed of nuclear genomes from two species, A. laterale (Blue Spotted Salamander) and A. jeffersonianum (Jefferson Salamander). We developed species-specific microsatellite markers for these two species as a tool for the identification and investigation of the genetic interactions between sexual and unisexual groups in areas where either sexual species is endangered or of special conservation concern (Ohio, Indiana, and Ontario). Keywords Ambystoma Microsatellites Mole salamanders Microsatellites were isolated as described in Kartzinel et al. (2012). Primers which amplified 144 loci (96 from two Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0320-7) contains supplementary material, which is available to authorized users. R. D. Denton (&) H. L. Gibbs Department of Evolution, Ecology, and Organismal Biology, Aronoff 300, The Ohio State University, 318 West 12th Avenue, Columbus, OH 43210, USA e-mail: [email protected] T. C. Glenn Department of Environmental Health Science, University of Georgia, Athens, GA 30602, USA
separate A. laterale libraries, 48 from an A. jeffersonianum library) and were initially screened across four individuals from each species. Those that showed amplification and polymorphism within a single species were then assayed in an additional 16 individuals sampled from across the species’ geographic range (Supplementary Table S1). PCR reactions for all primers were carried out in 10 ll reactions and consisted of 3.9 ll ddH20, 0.3 ll of mixed forward (CAG or M13) and reverse primer (untagged), 0.3 ll labelled M13R/CAG tag (6-FAM, HEX, or NED), 5 ll BioMixTM Red (BIOLINE), and 0.5 ll of template DNA. Primers were tested using the following temperature profile. First, each reaction was held at 95 °C for 2 min 30 s followed by 20 touchdown cycles (95 °C for 20 s, 60 °C for 20 s with a 0.5 °C decrease per cycle, and 72 °C for 30 s). The reaction was then subject to 15 cycles consisting of 95 °C for 20 s, 50 °C for 20 s, and 72 °C for 30 s. Finally, reactions were held at 72 °C for 10 min. PCR products were analyze on either a Applied Biosystems 3100 or 3730 DNA sequencer using a ROX-labeled internal size
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