title: Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA.
abstract: 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.
authors.affiliation: EcoGene®, Landcare Research, Auckland, New Zealand.
authors.forename: Ana
authors.initials: A
authors.lastname: Ramón-Laca
authors.affiliation: Institute for Applied Ecology, University of Canberra, Canberra, Australia.
authors.forename: Dianne
authors.initials: D
authors.lastname: Gleeson
authors.affiliation: Landcare Research, Lincoln, Canterbury, New Zealand.
authors.forename: Ivor
authors.initials: I
authors.lastname: Yockney
authors.affiliation: Landcare Research, Palmerston North, Manawatu, New Zealand.
authors.forename: Michael
authors.initials: M
authors.lastname: Perry
authors.affiliation: Landcare Research, Lincoln, Canterbury, New Zealand.
authors.forename: Graham
authors.initials: G
authors.lastname: Nugent
authors.affiliation: Arthur Rylah Institute for Environmental Research, Department of Environment and Primary Industries, Heidelberg, Victoria, Australia.
authors.forename: David M
authors.initials: DM
authors.lastname: Forsyth
mesh_terms: D000818:Animals; D001188:Artiodactyla / Q000145:classification* / Q000235:genetics*; D045303:Cytochromes b / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics; D005243:Feces; D008969:Molecular Sequence Data; D009520:New Zealand; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D011309:Preservation, Biological; D012335:RNA, Ribosomal / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D013045:Species Specificity
chemical_list: D004272:DNA, Mitochondrial; D012335:RNA, Ribosomal; C075526:RNA, ribosomal, 12S; D004247:DNA; D045303:Cytochromes b; D003576:Electron Transport Complex IV