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. Cross-species 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.
This is a preview of subscription content, log in via an institution to check access.
References
Biggs J, Ewald N, Valentini A, Gaboriaud C, Dejean T, Griffiths RA, Foster J, Wilkinson JW, Arnell A, Brotherton P, Williams P, Dunn F (2015) Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biol Conserv 183:19–28
Che J, Chen H-M, Yang J-X, Jin J-Q, Jiang K, Yuan Z-Y, Murphy RW, Zhang Y-P (2012) Universal COI primers for DNA barcoding amphibians. Mol Ecol Res 12:247–258
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299
Goldberg CS, Pilliod DS, Arkle RS, Waits LP (2011) Molecular detection of vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho giant salamanders. PLoS ONE 6:e22746
Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc Roy Soc B: Biol Sci 270:313–321
Pilliod DS, Goldberg CS, Arkle RS, Waits LP, Richardson J (2013) Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples. Can J Fish Aquat Sci 70:1123–1130
Rees HC, Bishop K, Middleditch DJ, Patmore JRM, Maddison BC, Gough KC (2014) The application of eDNA for monitoring of the Great Crested Newt in the UK. Ecol Evol 4:4023–4032
Smart AS, Tingley R, Weeks AR, van Rooyen AR, McCarthy MA (2015) Environmental DNA sampling is more sensitive than a traditional survey technique for detecting an aquatic invader. Ecol Appl 25:1944–1952
Smith MA, Poyarkov NA Jr, Hebert PDN (2008) CO1 DNA barcoding amphibians: take the chance, meet the challenge. Mol Ecol Res 8:235–246
Thomsen PF, Kielgast J, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012) Monitoring endangered freshwater biodiversity using environmental DNA. Mol Ecol 21:2565–2573
Vonesh JR, Mitchell JC, Howell K, Crawford AJ (2009) Rapid assessments of amphibian diversity. In: Dodd CK Jr (ed) Amphibian ecology and conservation. Oxford University Press, New York, pp 263–280
Acknowledgements
This work was funded by the Canada-Ontario Agreement concerning the Great Lakes Ecosystems (COA) and the Ontario Ministry of Natural Resources and Forestry (OMNRF).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
12686_2017_962_MOESM1_ESM.xlsx
Matrix of nucleotide substitutions (mismatches) among species at binding sites for each species’ forward primer, probe, and reverse primer. Rows = reference species; values listed in columns represent mismatches (forward primer/probe/reverse primer) in non-target species. Note that the matrices are not symmetrical, as the species-specific primers and probes target different amplicons within the COI barcoding segment. (XLSX 25 KB)
12686_2017_962_MOESM2_ESM.xlsx
Cross-species amplification results for frogs. Results show qPCR yields as copy number and cycle threshold (Ct) when amplifying 103 copies of the ~ 650 bp COI barcoding region using control DNA from non-target species. Dashes indicate no amplification occurred, and blank cells represent species that were not tested. (XLSX 16 KB)
12686_2017_962_MOESM3_ESM.xlsx
Cross-species amplification results for salamanders. Results show qPCR yields as copy number and cycle threshold (Ct) when amplifying 103 copies of the ~ 650 bp COI barcoding region using control DNA from non-target species. Dashes indicate no amplification occurred, and blank cells represent species that were not tested. (XLSX 18 KB)
Rights and permissions
About this article
Cite this article
Beauclerc, K., Wozney, K., Smith, C. et al. Development of quantitative PCR primers and probes for environmental DNA detection of amphibians in Ontario. Conservation Genet Resour 11, 43–46 (2019). https://doi.org/10.1007/s12686-017-0962-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12686-017-0962-3