An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (Canis rufus) experimental population area
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Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.
KeywordsCanis rufus Faecal DNA Genotyping errors Hybrid detection
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We thank Buddy Fazio, team leader of the Red Wolf Recovery Program, and the Recovery Implementation Team for continued support of our research efforts. Craig Miller, the Waits lab group and three anonymous reviewers provided helpful comments on this manuscript. Members of the red wolf field crew, Arthur Beyer, Chris Lucash, Scott McLellan, Michael Morse, Leslie Schutte, and Kathy Whidbee, and program volunteers assisted in collecting the scats. Bruce Creef and the ARNWR maintenance facility staff provided help with the ATVs. The US Department of Defense and Gary Melton, Wayne Daniels (AFBR), Harry Mann (NBR) allowed access to the bombing ranges. Debra Montgomery assisted with statistical analyses. Andrea Bristol, Jonathan Teeters and Melanie Murphy provided assistance in the laboratory. Funding was provided by the United States Fish and Wildlife Service.
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