Urine — a source for noninvasive genetic monitoring in wildlife

Abstract

Noninvasive samples are of increasing importance to study wild populations. In this study, we investigate the applicability of urine samples as the sole source of DNA for routine noninvasive genetic monitoring of wildlife using wolves (Canis lupus) as an example. Within the scope of a long‐term wolf population survey, we collected during winter snow tracking in Bieszczady Mountains, Poland 41 urine samples considered as utilizable for genetic analyses. DNA concentration was determined by quantitative real‐time polymerase chain reaction (qPCR) and six microsatellite loci were genotyped in threefold repeated genotyping experiments to assess the reliability of genetic analyses of urine. DNA concentration of 33 urine samples was successfully quantified and of 14 samples, we obtained congruent results for all analysed loci and all repeated genotyping experiments. The gender of urine samples was identified with a Y‐chromosome‐linked marker. Considering the high discovery rate of urine in conjunction with its genotype reliability, our study confirms that urine is a valuable source in noninvasive genetic monitoring. Additionally, preselection of samples via qPCR proved to be a powerful tool contributing to a beneficial cost‐value ratio of genetic analyses by minimizing genotyping errors.

Evidence of wolf dispersal in anthropogenic habitats of the Polish Carpathian Mountains

“…DNA from blood was puriWed either with a modiWcation of the phenol-chloroform procedure (Hogan et al 1986) or-in case of blood collected in snow-with the NucleoSpin ® Blood QuickPure Kit (Macherey-Nagel). Eight canine microsatellite loci (CPH2, CPH4, CPH8, CPH12, FH2096, FH2088, FH2079, C09.250; Hausknecht et al 2007;Lucchini et al 2002) previously applied to genetic wolf studies, were PCR ampliWed containing the following components (15 l volume): 1.5 mM (FH2096, FH2088, FH2079) or 3.0 mM MgCl 2 , respectively, 0.2 mM dNTPs, 1£ reaction buVer, 0.2 M of each primer, 0.6 U FirePol Taq DNA Polymerase (Solis Biodyne) and 1.8 l DNA. PCRs were performed in a BIOMETRA UNO II CYLER or in an EPPENDORF MASTER gradient cycler in 35 cycles under the conditions described by Hausknecht et al 2007 (annealing: CPH12 57°C, C09.250 58°C).…”

“…Eight canine microsatellite loci (CPH2, CPH4, CPH8, CPH12, FH2096, FH2088, FH2079, C09.250; Hausknecht et al 2007;Lucchini et al 2002) previously applied to genetic wolf studies, were PCR ampliWed containing the following components (15 l volume): 1.5 mM (FH2096, FH2088, FH2079) or 3.0 mM MgCl 2 , respectively, 0.2 mM dNTPs, 1£ reaction buVer, 0.2 M of each primer, 0.6 U FirePol Taq DNA Polymerase (Solis Biodyne) and 1.8 l DNA. PCRs were performed in a BIOMETRA UNO II CYLER or in an EPPENDORF MASTER gradient cycler in 35 cycles under the conditions described by Hausknecht et al 2007 (annealing: CPH12 57°C, C09.250 58°C). We determined the size of microsatellite alleles on an ALFExpress II DNA analyser (Amersham Pharmacia Biotech) using ALLELELINKS version 1.02 software (Amersham Pharmacia Biotech).…”

“…For sex identiWcation of the four blood samples collected in snow, we carried out multiplex PCRs containing a fragment of a canine microsatellite locus (ZuBeCa 65; described in Hausknecht et al 2007) as internal positive control and the MS41 locus indicating male sex (Olivier et al 1999;modiWed by Sundqvist et al 2001). PCR reactions (15 l) contained 2 mM MgCl 2 , 0.2 mM dNTPs, 1£ reaction buVer, 0.14 M of ZuBeCa65 primer and 0.4 M of MS 41 primer, 0.6 U FirePol Taq DNA Polymerase (Solis Biodyne) and 2.4 l DNA.…”

Confirmation of wolf residence in Northern Hungary by field and genetic monitoring

“…We applied snow-tracking procedures as described in Hausknecht et al (2007) and Gula (2008b). Track routes, wolf numbers, dens sites, urination and defecation sites were recorded with Global Positioning System (GPS) data loggers and scat and urine samples were collected for genetic analyses.…”

Faecal pellet size can be used to differentiate age-classes in caribou: implications for non-invasive genetic studies

“…Protocols have been recently developed to optimize the quality and quantity of DNA from source material including urine, hair and faeces (Ball et al 2007;Broquet et al 2007;Hausknecht et al 2007;Waits and Paetkau 2005). These techniques have enabled researchers to collect DNA from species that would be difficult to sample through traditional methods (blood, tissue).…”