Wildlife Research Wildlife Research Society
Ecology, management and conservation in natural and modified habitats
RESEARCH ARTICLE

Effect of sample age and season of collection on the reliability of microsatellite genotyping of faecal DNA

Maxine P. Piggott
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School of Biological Sciences, Monash University, Vic. 3800, Australia. Email: Maxine.Piggott@sci.monash.edu.au

Wildlife Research 31(6) 485-493 https://doi.org/10.1071/WR03096
Submitted: 10 October 2003  Accepted: 3 August 2004   Published: 13 December 2004

Abstract

Individual identification of animals from DNA in field-collected faecal samples is becoming an increasingly important tool in wildlife population monitoring. A major issue relevant to the application of this technique is the reliability of the genotypes obtained. I investigated the effect of sample age and season of collection on amplification rates and reliability of microsatellite genotypes amplified from faecal DNA of a marsupial herbivore, the brush-tailed rock-wallaby (Petrogale penicillata) and a eutherian carnivore, the red fox (Vulpes vulpes). Comparison of DNA profiles from 1 day to 6 months for both species suggests that as the age of the faeces increases there is less good-quality DNA present on the surface of the faeces, resulting in significantly decreasing amplification rates and increasing genotyping error rates over time. No microsatellite PCR products were obtained from samples older than 3 months from any faecal DNA extract in either season. For both species, faeces collected during the summer trial yielded high-quality DNA for up to one week. Faeces collected in winter had significantly lower amplification rates and higher genotyping errors than the summer-collected samples. Computer simulations were used to estimate the probability of obtaining false genotypes when genotyping faecal samples of various ages. These revealed that three replicates is sufficient to prevent identification of false individuals for P. penicillata from faeces up to one week old in both summer and winter but more replicates may be required for older samples, particularly in winter. In contrast, up to eight replicates may be required for fox faeces collected in winter, particularly if more than one week old. These results also suggest that it is difficult to visually identify faecal age for V. vulpes, and any study using fox faeces would need to account for the likely inclusion of older faeces in a field collection. For P. penicillata, faecal age could be accurately assessed, particularly when less than one week old and targeting faeces that match the two most reliable appearance classes described here would be an efficient sampling strategy. It is recommended that the appropriate PCR replication protocol for any given study should be tailored to the error rates expected for the oldest samples likely to be collected. This study is the first to thoroughly investigate the effects of sample age and season of collection on microsatellite genotyping from faecal samples and provides guidelines for sampling and PCR repetition strategies for field-based non-invasive DNA studies.


Acknowledgments

Thanks to the staff at Healesville Sanctuary and Keith Turnbull Research Institute, Frankston, for their assistance. Thanks to Gerry Quinn and Sam Banks for statistical advice. Sam Banks and Andrea Taylor are also thanked for comments on the manuscript. This research was funded by an Australian Research Council SPIRT grant and was carried out under Monash University Animal Ethics Permit No. BSCI/2000/09. M. Piggott is supported by an APAI Scholarship from an Australian Research Council SPIRT grant and this research was partly funded by the Victorian Government Department of Sustainability and Environment and Zoological Parks and Garden Board of Victoria.


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