Skip to main content
SpringerLink
Log in

Identification of a broad spectrum of mammalian and avian species using the short fragment of the mitochondrially encoded cytochrome b gene

  • Original Article
  • Published:
Forensic Science, Medicine and Pathology Aims and scope Submit manuscript

Abstract

Mitochondrial DNA (mtDNA), especially the gene for cytochrome b (MT-CYB), has been found to be highly informative for species identification. In this study, we present the results of the analysis of a 127 bp long fragment of MT-CYB, amplified using universal primers, variable enough to be used for species identification and discrimination, even in highly degraded animal samples. The total number of analyzed species in this study was 30, including 17 mammalian and 13 bird species. Using a newly created primer pair, we successfully amplified and sequenced the target sequence in almost all tested species. The amplification was incomplete in just two species, and as a result, partial, but still variable sequences, were obtained. Using the target fragment we successfully identified all tested samples. Initial results suggested that the intraspecies genetic diversity of the target region, in all tested species, was low – from 0 to 4.72%. The interspecies genetic diversity of the target region, crucial for successful discrimination, showed relatively high diversity, ranging from 8.36% to 42.52%. Given its short length, the target region should be used for species determination, particularly in samples that are degraded or are low in DNA quantity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Tobe SS, Linacre AMT. A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene. Electrophoresis. 2008;29:340–7.

    Article  CAS  PubMed  Google Scholar 

  2. Xi J, Zhu W, Zhou Y, Liu Z, Chen Y, Zhao Z. Identification of mammalian species using the short and highly variable regions of mitochondrial DNA. Mitochondrial DNA. 2015;26:550–4.

    Article  CAS  Google Scholar 

  3. Parson W, Pegoraro K, Niedersttter H, Foger M, Steinlechner M. Species identification by means of the cytochrome b gene. Int J Legal Med. 2000;114:23–8.

    Article  CAS  Google Scholar 

  4. Lopez-Oceja A, Gamarra D, Borragan S, Jimenez-Moreno S, de Pancorbo MM. New cyt b gene universal primer set for forensic analysis. Forensic Sci Int Genet. 2016;23:159–65.

    Article  CAS  PubMed  Google Scholar 

  5. Jun J, Han SH, Jeong TJ, Park HC, Lee B, Kwak M. Wildlife forensics using mitochodrial DNA sequences: species identification based on hairs collected in the field and confiscated tanned Felidae leathers. Genes Genom. 2011;33:721–6.

    Article  CAS  Google Scholar 

  6. Hsieh HM, Chiang HL, Tsai LC, Lai SY, Huang NE, Linacre A, et al. Cytochrome b gene for species identification of the conservation animals. Forensic Sci Int. 2001;122:7–18.

    Article  CAS  PubMed  Google Scholar 

  7. Melton T, Holland C. Routine forensic use of the mitochondrial 12s ribosomal RNA gene for species identification. J Forensic Sci. 2007;52:1305–7.

    CAS  PubMed  Google Scholar 

  8. Pfeiffer I, Burger J, Brening B. Diagnostic polymorphisms in the mitochondrial cytochrome b gene allow discrimination between cattle, sheep, goat, roe buck and deer by PCR-RFLP. BMC Genet. 2004;5:30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bravi CM, Liron JP, Mirol PM, Ripoli MV, Garcia-Peral P, Giovambattista G. A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. Legal Med. 2004;6:246–51.

    Article  CAS  PubMed  Google Scholar 

  10. Spychaj A, Mozdzaik EP, Pospiech E. PCR methods in meat species identification as a toll for the verification of regional and traditional meat products. Acta Sci Pol Technol Aliment. 2009;8:5–20.

    CAS  Google Scholar 

  11. Hsieh YHP, Sheu SC, Bridgman CR. Development of a monoclonal antibody specific to cooked mammalian meats. J Food Prot. 1998;61:476–81.

    Article  CAS  PubMed  Google Scholar 

  12. Fernandes CA, Ginja C, Pereira I, Tenreiro R, Bruford MW, Santos-Reis M. Species-specific mitochondrial DNA markers for identification of non-invasive samples from sympatric carnivores in the Iberian Peninsula. Conserv Genet. 2008;9:681–90.

    Article  CAS  Google Scholar 

  13. Girish PS, Anjaneyulu ASR, Viswas KN, Anand M, Rajkumar N, Shivakumar BM, et al. Sequence analysis of mitochondrial 12s RNA gene can identify meat species. Meat Sci. 2004;66:551–6.

    Article  CAS  PubMed  Google Scholar 

  14. Hebert PDN, Ratnasingham S, de Waard RJ. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci. 2003;270:96–9.

    Google Scholar 

  15. Hsieh HM, Tsai CC, Tsai LC, Chiang HL, Huang NE, Shih RTP, et al. Species identification of meat products using the cytochrome b gene. Forensic Sci J. 2005;4:29–36.

    Google Scholar 

  16. Imaizumi K, Akutsu T, Miyasaka S, Yoshino M. Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA. Int J Legal Med. 2007;121:184–91.

    Article  Google Scholar 

  17. Irwin DM, Kocher DT, Wilson CA. Evolution of the cytochrome b gene of mammals. J Mol Evol. 1991;32:128–44.

    Article  CAS  PubMed  Google Scholar 

  18. Kocher T, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, et al. Dynamics of mitochondrial DNA evolution in mammals: amplification and sequencing with conserved primers. Proc Natl Acad Sci U S A. 1989;86:6196–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mitani T, Akane A, Tokiyasu T, Yoshimura S, Okki Y, Yoshida M. Identification of animal species using the partial sequences in the mitochondrial 16s rRNA gene. Legal Med. 2009;11:449–50.

    Article  Google Scholar 

  20. Muangkram Y, Wajjwalku W, Amano A, Sukmak M. The novel primers for mammal species identification-based mitochondrial cytochrome b sequence: implication for reserved wild animals in Thailand and endangered mammal species in Southeast Asia. Mitochondrial DNA A. DNA Mapp Seq Anal. 2018;29:62–72.

    CAS  PubMed  Google Scholar 

  21. Naidu A, Fitak RR, Munguia-Vega A, Culver M. Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals. Mol Ecol Resour. 2012;12:191–6.

    Article  CAS  PubMed  Google Scholar 

  22. Verma SK, Singh L. Novel universal primers establish identity of an enormous number of animal species for forensic application. Mol Ecol Notes. 2003;3:28–31.

    Article  CAS  Google Scholar 

  23. Morley JM, Bark JE, Evans CE, Perry JG, Hewitt CA, Tully G. Validation of mitochondrial DNA minisequencing for forensic casework. Int J Legal Med. 1999;12:241–8.

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to express their gratitude to colleagues from a private DNA Center for Genetics laboratory in Belgrade, and colleagues from the University of Belgrade Faculty of Biology, for providing the samples.

Author information

Authors and Affiliations

  1. DNA Centre for Genetics, Belgrade, 11000, Serbia

    Marko Andrejevic

  2. Faculty of Biology, Center for Forensic and Applied Molecular Genetics, University of Belgrade, Studentski trg 3, Belgrade, 11000, Serbia

    Milica Keckarevic Markovic, Milica Mihajlovic, Vanja Tanasic, Miljana Kecmanovic & Dusan Keckarevic

  3. Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, 11000, Serbia

    Biljana Bursac

Authors
  1. Marko Andrejevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Milica Keckarevic Markovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Biljana Bursac
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Milica Mihajlovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vanja Tanasic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Miljana Kecmanovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dusan Keckarevic
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Milica Keckarevic Markovic.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in this study were in accordance with the ethical standards of the Serbian national research committee and with the 1964 Helsinki declaration and its later amendments. The study was approved by authorities of Faculty of Biology at the University of Belgrade.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Andrejevic, M., Markovic, M.K., Bursac, B. et al. Identification of a broad spectrum of mammalian and avian species using the short fragment of the mitochondrially encoded cytochrome b gene. Forensic Sci Med Pathol 15, 169–177 (2019). https://doi.org/10.1007/s12024-019-00096-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12024-019-00096-4

Keywords

Search

Navigation