Development of quantitative PCR primers and probes for environmental DNA detection of amphibians in Ontario
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TECHNICAL NOTE
Development of quantitative PCR primers and probes for environmental DNA detection of amphibians in Ontario Kaela Beauclerc1 · Kristyne Wozney2 · Caleigh Smith2 · Chris Wilson2 Received: 30 August 2017 / Accepted: 22 December 2017 © Crown 2018
Abstract DNA from environmental samples (eDNA) is increasingly being used to detect and monitor elusive species. We developed species-specific eDNA primers and probes for qPCR detection of 24 amphibian species native to Ontario, Canada. Crossspecies testing confirmed their high specificity and low cross-species amplification, as well as their ability to detect DNA from target species at low concentrations. These detection tools should prove useful for monitoring at-risk amphibian species throughout their native ranges. Keywords Amphibian · eDNA · Quantitative PCR Lack of accurate data on amphibian distributions can hinder effective conservation and management. Traditionally, amphibian surveys involve capture of organisms via nets or traps, call surveys, visual encounter surveys or a combination of these (Vonesh et al. 2009). Intensive capture-based surveys risk injuring specimens and/or disrupting habitats, and survey success is often dependent on the skill of field crews in identifying species by call or appearance (Vonesh et al. 2009). Environmental DNA (eDNA) surveys have been established as sensitive, non-invasive and cost-effective tools for monitoring aquatic species including invasive and endangered amphibians (Biggs et al. 2015; Goldberg et al. 2011; Rees et al. 2014), and eDNA has been shown to be more sensitive than traditional sampling to document habitat occupancy by amphibians (Pilliod et al. 2013; Smart et al. 2015; Thomsen et al. 2012). Here we describe the development and optimization of species-specific eDNA primer and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12686-017-0962-3) contains supplementary material, which is available to authorized users. * Kristyne Wozney [email protected] 1
Wildlife Research and Monitoring Section, Ontario Ministry of Natural Resources and Forestry, Trent University, 2140 East Bank Drive, Peterborough, ON K9L 0G2, Canada
Aquatic Research and Monitoring Section, Ontario Ministry of Natural Resources and Forestry, Trent University, 2140 East Bank Drive, Peterborough, ON K9L 0G2, Canada
2
probe sets for 24 of Ontario’s native amphibian species for use in monitoring. Reference samples of target species were obtained as frozen or ethanol-preserved tissue from the Royal Ontario Museum (ROM), University of Guelph, and Laurentian University, or nonlethal toe clips from field specimens (Trent University animal care permit 23907). Genomic DNA was extracted using the E.Z.N.A. Tissue DNA kit (Omega Biotek) and eluted in 100–200 μL of TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0). All specimens were amplified and sequenced at the COI barcoding region using universal primers (Che et al. 2012; Folmer et al. 1994; Hebert et al. 2003; Smith et al. 2008)
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