A novel method to optimise the utility of underused moulted plumulaceous feather samples for genetic analysis in bird conservation

From Springer Nature via Jisc Publications RouterHistory: received 2019-04-05, accepted 2019-10-17, registration 2019-10-18, epub 2019-10-24, online 2019-10-24, ppub 2020-09Publication status: PublishedFunder: University of ChesterAbstract: Non-invasive sampling methods are increasingly being used in conservation research as they reduce or eliminate the stress and disturbance resulting from invasive sampling of blood or tissue. Here we present a protocol optimised for obtaining usable genetic material from moulted plumulaceous feather samples. The combination of simple alterations to a ‘user-developed’ method, comprised of increased incubation time and modification of temperature and volume of DNA elution buffer, are outlined to increase DNA yield and significantly increase DNA concentration (W = 81, p < 0.01, Cohens’s d = 0.89). We also demonstrate that the use of a primerless polymerase chain reaction (PCR) technique increases DNA quality and amplification success when used prior to PCR reactions targeting avian mitochondrial DNA (mtDNA). A small amplicon strategy proved effective for mtDNA amplification using PCR, targeting three overlapping 314–359 bp regions of the cytochrome oxidase I barcoding region which, when combined, aligned with target-species reference sequences. We provide evidence that samples collected non-invasively in the field and kept in non-optimal conditions for DNA extraction can be used effectively to sequence a 650 bp region of mtDNA for genetic analysis