Abstract
We evaluated PCR primer sets to determine the most effective technique for identifying sex of northern ungulates. We sought markers that required only a single pair of primers to amplify both X- and Y-linked alleles; that amplified X- and Y-linked products that were easily distinguishable using agarose gel electrophoresis; and that produced short amplicons amenable to amplification using DNA of poor quality and low quantity, as is often found in non-invasively collected samples such as feces. Primer pairs KY1/KY2 and SE47/SE48, which amplify X- and Y-specific alleles of the amelogenin gene, met our criteria and were tested for moose (Alces alces), mountain goat (Oreamnos americanus), Sitka black-tailed deer (Odocoileus hemionus sitkensis), and caribou (Rangifer tarandus). KY primers amplified shorter PCR products than did SE primers; moreover, SE primers inconsistently amplified certain Y-chromosome products, creating potential for misidentification of sex. DNA fragments amplified using KY primers were sequenced for each species, allowing us to characterize a 45-bp deletion for Y-linked alleles (136-bp product) relative to X-linked alleles (181-bp product) in all species and a 9-bp deletion in the X-linked allele of moose relative to other species. This is the first sex-determination technique using PCR reported for several ungulate species of Alaska. Although other protocols exist for cervids and bovids, this is the first report of markers meeting the aforementioned criteria for Odocoileus, the most abundant and intensively managed genus of large mammals in North America.
References
Ball MC, Pither R, Manseau M, Clark J, Petersen SD, Kingston S, Morrill N, Wilson P (2007) Characterization of target nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template. Conserv Genet 8:577–586. doi:10.1007/s10592-006-9193-y
Barnes WM (1992) The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene 112:29–35. doi:10.1016/0378-1119(92)90299-5
Brinkman TJ, Kofinas GP, Chapin FSIII, Person DK (2007) Influence of hunter adaptability on resilience of subsistence hunting systems. J Ecol Anthro 11:58–63
Buchan JC, Archie EA, VanHorn RC, Moss CJ, Alberts SC (2005) Locus effects and sources of error in noninvasive genotyping. Mol Ecol Notes 5:680–683. doi:10.1111/j.1471-8286.2005.01002.x
Cooper DL, Baptist EW (1991) Degenerate oligonucleotide sequence-directed cross-species PCR cloning of the BCP 54/ALDH 3 cDNA: priming from inverted repeats and formation of tandem primer arrays. PCR Methods Appl 1:57–62
Demarais S, Krausman PR (2000) Ecology and management of large mammals in North America. Prentice Hall, Upper Saddle River, New Jersey
Ennis S, Gallagher TF (1994) A PCR-based sec-determination assay in cattle based on the bovine amelogenin locus. Anim Genet 25:425–427
Lindsay AR, Belant JL (2007) A simple and improved PCR-based technique for white-tailed deer (Odocoileus virginianus) sex identification. Conserv Genet 9:443–447. doi:10.1007/s10592-007-9326-y
Maudet C, Luikart G, Dubray D, Von Hardenberg A, Taberlet P (2004) Low genotyping error rates in wild ungulate feces sampled in winter. Mol Ecol Notes 4:772–775. doi:10.1111/j.1471-8286.2004.00787.x
Mazza R (2003) Hunter demand for deer on Prince of Wales Island, Alaska: an analysis of influencing factors. General Technical Report PNW-GTR–581, USDA forest service. Pacific Northwest Research Station, Portland
Murphy MA, Waits LP, Kendall KC (2003) Influence of diet on faecal DNA amplification and sex identification in brown bears (Ursus arctos). Mol Ecol 12:2261–2265. doi:10.1046/j.1365-294X.2003.01863.x
Neeser D, Liechti-Gallati (1995) Sex determination of forensic samples by simultaneous PCR amplification of α-satellite DNA from both the X and Y chromosomes. J Forensic Sci 40:239–241
Palsbøll PJ, Vader A, Bakke I, Rafaat El-Gewely M (1992) Determination of gender in cetaceansby the polymerase chain reaction. Can J Zool 70:2166–2170. doi:10.1139/z92-292
Pfeiffer I, Brenig B (2005) X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European red deer (Cervus elaphus). BMC Genet 6:16. doi:10.1186/1471-2156-6-16
Pomp D, Medrano JF (1991) Organic solvents as facilitators of polymerase chain reaction. Biotechniques 10:58–59
Reynolds R, Varlaro J (1996) Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences. J Forensic Sci 41:279–286
Sefc KM, Payne RB, Sorenson MD (2003) Microsatellite amplification from museum feather samples: effects of fragment size and template concentration of genotyping errors. Auk 120:982–989. doi:10.1642/0004-8038(2003)120[0982:MAFMFS]2.0.CO;2
Shaw CN, Wilson PJ, White BN (2003) A reliable molecular method of gender determination for mammals. J Mammal 84:123–128. doi:10.1644/1545-1542(2003)084<0123:ARMMOG>2.0.CO;2
Sullivan KM, Mannucci A, Kimpton CP, Gill P (1993) A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques 15:636–641
Waits LP, Paetkau D (2005) Noninvasive genetic sampling of wildlife. J Wildl Manage 69:1419–1433. doi:10.2193/0022-541X(2005)69[1419:NGSTFW]2.0.CO;2
Wang L-F, Rakela J, Laskus T (1997) Head-to-tail primer tandem repeats generated in hemi-nested PCR. Mol Cell Probes 11:385–387. doi:10.1006/mcpr.1997.0122
Weikard R, Pitra C, Kuhn C (2006) Amelogenin cross-amplification in the family Bovidae and its application for sex determination. Mol Reprod Dev 73:1333–1337. doi:10.1002/mrd.20554
Yamauchi K, Hamasaki S, Miyazaki K, Kikusui T, Takeuchi Y, Mori Y (2000) Sex determination based on fecal DNA analysis of the amelogenin gene in sika deer (Cervus nippon). J Vet Med Sci 62:669–671. doi:10.1292/jvms.62.669
Acknowledgments
Funding was provided by Alaska Department of Fish and Game Division of Wildlife Conservation, the Alaska Trappers Association, the National Science Foundation’s IGERT (Resilience and Adaptation) and LTER Programs, the University of Alaska Fairbanks and Institute of Arctic Biology, and the USDA Forest Service. We thank P. Barboza, L. Dickerson, B. Porter, D. Person, and K. White for assistance collecting and donating tissue and blood samples. Laboratory assistance during our study was provided by N. Swensgard and K. Colson.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Brinkman, T.J., Hundertmark, K.J. Sex identification of northern ungulates using low quality and quantity DNA. Conserv Genet 10, 1189–1193 (2009). https://doi.org/10.1007/s10592-008-9747-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10592-008-9747-2