TECHNICAL NOTE TaqMan assays for species identification of the red squirrel (Sciurus vulgaris) and the grey squirrel (Sciurus carolinensis) Denise B. O’Meara • Peter D. Turner • Lee Coffey • Catherine O’Reilly Received: 23 June 2011 / Accepted: 9 January 2012 Ó Springer Science+Business Media B.V. 2012 Abstract We have developed TaqMan based assays for species-specific identification of two species of squirrel found in the British Isles, the native red squirrel (Sciurus vulgaris) and the introduced north American grey squirrel (Sciurus carolinensis). These assays correctly identified tissue and hair samples of both species and there was no cross-species amplification. This is a useful method for non-invasive surveys to help conserve the red squirrel and manage the spread of the grey squirrel in the UK and Ireland. Keywords Real-time PCR  Non-invasive  Hair-tubes  Mitochondrial DNA Hair-tubes are inexpensive to construct and easy to install, and can potentially provide distribution information on squirrel populations (Gurnell et al. 2001). However, inter- pretation is limited by the possibility of more than one species depositing a hair sample and by the variations in hair colour found in both red and grey squirrels. Correct identification of samples is important as the species are found to overlap in range prior to the red squirrel being outcompeted. Over recent years, non-invasive genetic sampling has been used to census populations (Mullins et al. 2010). Real-time PCR has been successfully used to species-type fox (Vulpes vulpes), pine marten (Martes martes), wood mouse (Apodemus sylvaticus), bank vole (Clethrionomys glareolus), common shrew (Sorex aran- eus), pygmy shrew (Sorex minutus) and water shrew (Neomys fodiens) (Moran et al. 2008; O’Reilly et al. 2008; Mullins et al. 2010). The red squirrel is of conservation concern in the UK and Ireland, due to the presence the North American grey squirrel, an introduced competitor (Finnegan et al. 2008). We have developed two TaqMan assays capable of identifying red and grey squirrel. Red and grey squirrel tissue and hair samples from both Ireland and the UK were used to assess the geographic robustness of the assays (Table 1). Genomic DNA was extracted from tissue samples using the Zymo ZR Genomic DNA II kit (Zymo Research) used according to the man- ufacturer’s instructions. DNA was extracted from hair as described in Mullins et al. (2010). Two species-specific real-time polymerase chain reaction (PCR) TaqMan TM MGBÒ probe assays were designed to target a short region of mitochondrial D-loop DNA using haplo- types AF1110001–AF111027 (Barratt et al. 1999) and AM412650–AM412675 (Finnegan et al. 2008). Sequences were aligned using MegAlign 5.05 (DNASTAR) and con- served species-specific regions were identified and used to design primers and probes using Primer Express 2 software (Applied Biosystems), targeting species-specific nucleotide polymorphisms towards the 3 0 ends of the primers to enhance the specificity and sensitivity of the amplification. Primers and fluorescently labelled probes were purchased from MWG-Biotech AG (Table 2). PCR with TaqMan MGB labelled probes consisted of 5 ll TaqMan Universal PCR Master Mix (Applied Bio- systems), 100 nmol each primer, 100 nmol probe and 1 ll DNA (dilution determined empirically) in a total volume of 10 ll. Negative controls contained molecular grade water instead of DNA. All PCR reactions were carried out in an ABI 7300 real-time PCR system with MicroAmp Optical 96-well reaction plates (Applied Biosystems) and data was analysed using version 2.2.1 of the SDS software (Applied D. B. O’Meara (&)  P. D. Turner  L. Coffey  C. O’Reilly Department of Chemical and Life Sciences, Waterford Institute of Technology, Cork Road, Waterford, Ireland e-mail: domeara@wit.ie 123 Conservation Genet Resour DOI 10.1007/s12686-012-9602-0