Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples

Abstract

The single lynx species present in the Iberian Peninsula—Lynx pardinus (Temmink, 1827)—is one of the most threatened felines in the world so, any biological indication of its presence is significant. However, it is a very elusive species and physical evidence through direct visualization or camera trapping is difficult to obtain. Genetic noninvasive methods, in particular, molecular scatology, are a reliable alternative to unequivocally identify the presence of a species in a given geographical area. We have developed a highly specific and sensitive molecular method to identify the presence of this critically endangered lynx species from severely deteriorated fecal samples. The method consists in a double specific nested PCR of mitochondrial D-loop sequences and two diagnostic SNPs detected by a multiplex primer extension reaction.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

References

  1. Harayama Y, Oki T, Hayashi T, Tsukada K, Ota M, Asamura H (2011) A comparative study on the techniques of SNPs analysis for degraded DNA samples. Forensic Sci Int Genet Supp Ser 3(1):e180–e181

    Article  Google Scholar 

  2. Kitpipit T, Tobe SS, Kitchener AC, Gill P, Linacre A (2012) The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification—Implications for forensic purposes. Forensic Sci Int Genet 6(2):250–257

    Article  CAS  PubMed  Google Scholar 

  3. Kitpipit T, Thongjued K, Penchart K, Ouithavon K, Chotigeat W (2017) Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin. Forensic Sci Int Genet 27:106–115

    Article  CAS  PubMed  Google Scholar 

  4. Mukherjee N, Mondol S, Andheria A, Ramakrishnan U (2007) Rapid multiplex PCR based species identification of wild tigers using non-invasive samples. Conserv Genet 8(6):1465–1470

    Article  CAS  Google Scholar 

  5. Mumma MA, Zieminski C, Fuller TK, Mahoney SP, Waits LP (2015) Evaluating noninvasive genetic sampling techniques to estimate large carnivore abundance. Mol Ecol Resour 15(5):1133–1144

    Article  PubMed  Google Scholar 

  6. Palomares F, Godoy JA, Píriz A, O’Brien SJ (2002) Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx. Mol Ecol 11(10):2171–2182

    Article  CAS  PubMed  Google Scholar 

  7. Pires AE, Fernandes ML (2003) Last lynxes in Portugal? Molecular approaches in a pre-extinction scenario. Conserv Genet 4(4):525–532

    Article  CAS  Google Scholar 

  8. Stenglein JL, De Barba M, Ausband DE, Waits LP (2010) Impacts of sampling location within a faeces on DNA quality in two carnivore species. Mol Ecol Resour 10(1):109–114

    Article  CAS  PubMed  Google Scholar 

  9. Taberlet P, Waits LP, Luikart G (1999) Noninvasive genetic sampling: look before you leap. Trends Ecol Evol 14(8):323–327

    Article  CAS  PubMed  Google Scholar 

  10. Waits LP, Paetkau D (2005) Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. J Wildl Manag 69(4):1419–1433

    Article  Google Scholar 

Download references

Acknowledgements

This work was funded by the Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente (MAGRAMA), Project 16MNSV002. Thanks are due to the Center El Acebuche in the Doñana National Park (MAGRAMA - Junta de Andalucía) for providing fecal samples from animals bred in captivity, and to Dr. José Antonio Dávila (Instituto de Investigación en Recursos Cinegéticos-Universidad de Castilla La Mancha, IREC-UCLM) and Dr. Eva Martinez Nevado (Zoo-Aquarium, Madrid) for kindly providing additional lynx samples.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Pilar Arana.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Alejandra Cruz and Beatriz Matallanas have equally contributed to the article.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cruz, A., Matallanas, B., Lobón-Rovira, J. et al. Double specific nested PCR and diagnostic SNP assay for species identification in lynx fecal critical samples. Conservation Genet Resour 11, 173–175 (2019). https://doi.org/10.1007/s12686-018-0993-4

Download citation

Keywords

  • Iberian lynx
  • Fecal DNA
  • Threatened species
  • Nested PCR
  • Diagnostic SNPs