Development of a new faecal DNA extraction method (HV-CTAB-PCI) for amplification of mitochondrial and nuclear markers used in genetic analyses of dugongs (Dugong dugon)

Abstract

Non-invasively collected faecal samples are an alternative source of DNA to tissues, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to develop a new tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, amplification success of genetic markers from dugong faeces was compared between an established QIAamp and a newly developed DNA extraction method. Faecal DNA extracted using a new 'High Volume-CTAB-PCI' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of dugong skin DNA. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified mtDNA and nDNA better than eroded scats. Using the HV-CTAB-PCI method, nDNA was successfully amplified for the first time from dugong faeces. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.