Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA

• Published in: PloS one 2014-03, Vol.9 (3), p.e92043
• Main Authors: Ramón-Laca, Ana, Gleeson, Dianne, Yockney, Ivor, Perry, Michael, Nugent, Graham, Forsyth, David M
• Format: Article
• Language: English
• Subjects: Alpine environments; Analysis; Animals; Artiodactyla; Artiodactyla - classification; Artiodactyla - genetics; Biology and Life Sciences; Cervidae; Conservation; Cytochrome; Cytochromes b - genetics; Deer; Deoxyribonucleic acid; DNA; DNA - analysis; DNA - genetics; DNA, Mitochondrial - genetics; Ecological monitoring; Ecology and Environmental Sciences; Electron Transport Complex IV - genetics; Feces; Genes; Genetic analysis; High resolution; Hybridization; Identification; Mammals; Meat industry; Melting; Methods; Mitochondrial DNA; Molecular Sequence Data; Multiplexing; New Zealand; Nucleic Acid Denaturation - genetics; Pests; Polymerase chain reaction; Preservation, Biological; Primers; Reference Standards; Reproducibility of Results; RNA; RNA, Ribosomal - genetics; rRNA 12S; Sheep; Species; Species Specificity; Strategic management; Studies; White-tailed deer; Wildlife conservation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0092043

Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA.