Abstract
We have developed new specific primers for sex determination from forensic samples of wolves (Canis lupus), such as hair, saliva, faecal, tooth and urine samples. In order to improve molecular sexing, we performed a multiplex semi-nested polymerase chain reaction (PCR) and several replicated amplifications per sample to avoid errors in low quantity DNA samples, such as allelic dropout and false alleles. The sex of individuals is automatically determined by capillary electrophoresis with a fluorescently labelled internal sex-specific primer from each pair. Our method yielded sex identification on 100% of invasive samples and 93% of forensic samples, being one of the highest success rates obtained from wild animals.
References
Amills M, Vidal O, Varona L, Tomàs A, Gil M, Sánchez A, Noguera JL (2005) Polymorphism of the pig 2,4-dienoyl CoA reductase 1 gene (DECR1) and its association with carcass and meat quality traits. J Anim Sci 83:493–498
Bellemain E, Taberlet P (2004) Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces. Mol Ecol 4:519–522
Bellemain E, Nawaz MA, Valentini A, Swenson JE, Taberlet P (2007) Genetic tracking of the brown bear in northern Pakistan and implications for conservation. Biol Conserv 134:537–547
Bérubé M, Palsbǿll PJ (1996) Identification of sex in cetaceans by multiplexing with three ZFX and ZFY specific primers. Mol Ecol 5:283–287
Dallas JF, Coxon KE, Sykes T, Chanin PRF, Marshall F, Carss DN, Bacon PJ, Piertney SB, Racey PA (2003) Similar estimates of population genetic composition and sex ratio derived from carcasses and faeces of Eurasian otter Lutra lutra. Mol Ecol 12:275–282
Dewoody J, Nason JD, Hipkins VD (2006) Mitigating scoring errors in microsatellite data from wild populations. Mol Ecol 6:951–957
Durnin ME, Palsbǿll PJ, Ryder OA, McCullough DR (2007) A reliable genetic technique for sex determination of giant panda (Ailuropoda melanoleuca) from non-invasively collected hair samples. Conserv Genet 8:715–720
Hellborg L, Ellegren H (2003) Y chromosome conserved anchored tagged sequences (YCATS) for the analysis of mammalian male-specific DNA. Mol Ecol 12:289–291
Iudica CA, Whitten WM, Williams NH (2001) Small bones from dried mammal museum specimens as a reliable source of DNA. Biotechniques 30:732–736
Kohn MH, York EC, Kamradt DA, Haught G, Sauvajot RM, Wayne RK (1999) Estimating population size by genotyping faeces. Proc R Soc Lond 266:657–663
Lucchini E, Fabbri E, Marucco F, Ricci S, Boitani L, Randi E (2002) Noninvasive molecular tracking of colonizing wolf (Canis lupus) packs in the western Italian Alps. Mol Ecol 11:857–868
Mucci N, Randi E (2007) Sex identification of Eurasian otter (Lutra lutra) non-invasive DNA samples using ZFX/ZFY sequences. Conserv Genet 8:1479–1482
Murakami H, Yakamoto Y, Yoshimote K, Toshiaki O, Okamoto O, Shigeta Y, Doi Y, Miyaishi S, Ishizu H (2000) Forensic study of sex determination using PCR on teeth samples. Acta Med Okayama 54(1):21–32
Pang BCM, Cheung BKK (2007) Double swab technique for collecting touched evidence. Legal Med 9:181–184
Pfeiffer I, Völkel I, Täubert H, Brenig B (2004) Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification. Forensic Sci Int 141:149–151
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory Press, New York
Scandura M (2005) Individual sexing and genotyping from blood spots on the snow: a reliable source of DNA for non-invasive genetic surveys. Conserv Genet 6:871–874
Scandura M, Capitani C, Iacolina L, Marco A (2006) An empirical approach for reliable microsatellite genotyping of wolf DNA from multiple non-invasive sources. Conserv Genet 7:813–823
Seddon JM (2005) Canid-specific primers for molecular sexing using tissue or non-invasive samples. Conserv Genet 6:147–149
Smith DA, Ralls K, Hurt A, Adams B, Parker M, Maldonados E (2006) Assesing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses. Mol Ecol 15:387–406
Spriggs HF, Holmes NG, Breen M, Deloukas P, Langford CF, Ross MT, Carter NP, Davis ME, Knights C, Smith A, Farr CJ, McCarthy LC, Binns MM (2003) Construcion and integration of radiation-hybrid and cytogenetic maps of dog Chromosome X. Mamm Genome 14:214–221
Sugimoto T, Nagata J, Aramilev VV, Belozor A, Higashi S, McCullough DR (2006) Species and sex identification from faecal samples of sympatric carnivores, Amur leopard and Siberian tiger, in the Russian Far East. Conserv Genet 7:799–802
Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194
Ulrich PP, Romeo JM, Daniel LJ, Vyas GN (1993) An improved method for the detection of hepatitis C virus RNA in plasma utilizing heminested primers and internal control RNA. PCR Methods Appl 2:241–249
Valière N, Taberlet P (2000) Urine collected in the field as a source of DNA for species and individual identification. Mol Ecol Notes 9:2150–2152
Valière N, Bonenfant C, Toïgo C, Luikart G, Gaillard JM, Klein F (2007) Importance of a pilot study for non-invasive genetic sampling: genotyping errors and population size estimation in red deer. Conserv Genet 8:69–78
Zhan X, Li M, Zhang Z, Goossens B, Chen Y, Wang H, Bruford MW, Wei F (2006) Molecular censusing doubles giant panda population estimate in a key nature reserve. Curr Biol 16:R451–R452
Acknowledgements
We are grateful to the personnel from the Central Forest National Reserve (Zapovednik, Russia) for supplying tissue and tooth samples from wolves. We thank “Servei de Protecció de la Fauna, Flora i Animals de Companyia”, “Area d’ Activitats Cinegètiques”, “Guardes de Reserva de Fauna”, “Parc Natural del Cadí-Moixerò” “Forestal Catalana S.A” and “Servei de Diagnòstic Serològic de Leishmania - Servei d’Anàlisi de Fàrmacs, UAB” for making available faecal, urine and hair samples from wolves and dogs. Thanks are due also to Gary Walker for linguistic revision.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sastre, N., Francino, O., Lampreave, G. et al. Sex identification of wolf (Canis lupus) using non-invasive samples. Conserv Genet 10, 555–558 (2009). https://doi.org/10.1007/s10592-008-9565-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10592-008-9565-6