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.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…There are three reasons why researchers may want to sequence DNA from such samples: (1) to determine to which species the faeces belong, for example, as a mechanism to identify ungulate species (Ramón-Laca et al 2014); (2) because they want to determine the diet of the depositing animal (Vestheim & Jarman 2008;Shehzad et al 2012;Boyer et al 2013); or (3) because they are using the feeding ecology of the depositor as a way of sampling the biodiversity in its environment (Kuch et al 2002). This third approach envisions depositors as 'environmental samplers' and in effect faeces are 'biodiversity capsules' containing concentrated DNA from taxa consumed by the depositing species.…”

Extraction of DNA from captive‐sourced feces and molted feathers provides a novel method for conservation management of New Zealand kiwi (Apteryxspp.)

“…Furthermore, data retrieved from the genetic monitoring tool could supplement and/or verify data obtained from other monitoring methods such as geographical and call‐based individual identification methods already established for Apteryx mantelli and A. haastii (Corfield, Gillman, & Parsons, ; Dent & Molles, ). Our proposed protocol for collecting kiwi feces and sample collection for optimal DNA preservation is similar to those used in other surveys in New Zealand (Appendix (Ramón‐Laca et al., )), so presumably, its uptake by conservation officers would be relatively straightforward. Moreover, the noninvasive methodology developed here may well be transferable to other cryptic and hard‐to‐monitor bird species facing similar conservation challenges.…”

“…Twenty-five fresh fecal samples (≤1 day old) that had been protected from rainfall were collected from three captive-breeding facilities: Westshore Wildlife Reserve, Napier (location 1); Kiwi Encounter, Rainbow Springs, Rotorua (location 2); and Auckland Zoo, Auckland (location 3). A rayon swab was used to collect fecal material ( Figure 3b) from the surface of the stool (see instructional video: http://youtu.be/zniEFYLSgOI) (Bosnjak, Stevanov-Pavlovic, & Vucicevic, 2013;Ramón-Laca et al, 2014), and immediately on collection, the head of the swab was preserved in ca. 500 μl of lysis buffer (Longmire, Maltbie, & Baker, 1997) to avoid any biotic, hydrolytic, enzymatic, or microbial degradation that could cause ADO.…”

Progress and challenges associated with halal authentication of consumer packaged goods

“…26 One of the major challenges associated with highly processed food matrices is template concentration and DNA degradation, especially when the products are subjected to various chemical and thermal treatments. 27 Thus, to ensure successful PCR amplification, several factors such as copy number of the target gene, amplicon size and primer specificity should be considered, as they play a pivotal role in the subsequent amplification procedure. Recently, a novel double-gene detection method targeting mitochondrial DNA with two short amplicons (73 and 146 bp) has been evaluated on frankfurter products.…”