Forensic Science International: Genetics Supplement Series 2 (2009) 306–307

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/FSIGSS

Research article

Cytochrome b or cytochrome c oxidase subunit I for mammalian species

identification—An answer to the debate
Shanan S. Tobe a,*, Andrew Kitchener b, Adrian Linacre a
a
Centre of Forensic Science, WestCHEM, Department of Pure and Applied Chemistry, University of Strathclyde, 204 George Street, Glasgow, G1 1XW, UK
b
National Museums Scotland, Chambers Street, Edinburgh, EH1 1JF, UK

A R T I C L E I N F O A B S T R A C T

Article history: Species identification for forensic purposes is being increasingly used, as the value of non-human
Received 13 August 2009 evidence is realized. This requires the identification of the species before individual analysis can take
Accepted 14 August 2009 place. Traditionally the cytochrome b (cyt b) gene was used for species identification, but in 2003 the
cytochrome c oxidase subunit 1 (CO1) gene was introduced under the terminology ‘barcoding’. This
Keywords: started an ongoing debate as to which gene offers the best template for species identification (high inter-
Species identification species variability and low intra-species variation). Sequence data from 236 mammals were compared
Mammals
with multiple sequence alignments for a large number of human, cow and dog samples. Comparisons
Cytochrome b (cyt b)
Cytochrome c oxidase subunit 1 (COI)
were made based on the number of inter-species variations between the different species and the intra-
Inter-species variation species variation between members of the same species.
Intra-species variation ß 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction sequences. There are no set guidelines for what percentage

The prevalence of papers pertaining to non-human forensic
evidence is increasing as the value of non-human evidence is
realized. As each case is different, with different non-human
samples potentially present in different locations and at different
times of the year, new tests are being developed almost on a case
by case basis. As such there are very few tests that can analyze
animal DNA in the same manner that human DNA is analyzed.
There are commercial STR tests available for cats, dogs and some
livestock. Generally tests are based on sequence information from
a mitochondrial gene as mitochondrial DNA is available in trace
samples such as hair that may be present. The added benefit of
mitochondrial DNA is that it is possible to design universal primers
able to amplify a large range of species.
Initially, to identify animal samples to species level for forensic
purposes, tests were adapted from phylogeny studies; mainly
those of Kocher et al. [1] and Irwin et al. [2]. These tests required
the amplification of a section of the cytochrome b (cyt b) gene on
the mitochondrial genome. These assays based on the cyt b gene
were widely used throughout forensic science (e.g. Ref. [3]). The
assays use universal primers to amplify a section of the cyt b gene
(generally around 400 base pairs (bp)) which is then compared to a
reference sequence or, more recently, to an online database of
* Corresponding author.
E-mail address: shanan.s.tobe@strath.ac.uk (S.S. Tobe).
variation constituted an exclusion or inclusion.
In 2003, Hebert et al. [4] brought forth the idea of DNA
‘barcodes’ for species identification and taxonomy. Barcoding is
just a pseudonym for standard sequencing and is based on the
cytochrome oxidase subunit I gene (COI) on the mitochondrial
genome. The technique described also uses universal primers to
amplify approximately a 650 bp region of the COI gene. The 2003
paper describes 100% success when using this technique with
Lepidoptera [4]. For forensic science the ‘barcode’ is also compared
to a reference sequence or an online database of sequences. Again
there are no guidelines as to what constitutes an exclusion or
inclusion.
The use of cyt b or COI sparked a debate as to whether or not
barcoding was suitable for determining species [5] and since then
alternate genes such as the 16S rRNA [6] and ND1 [7] have been
suggested. Here we provide a direct comparison between the cyt b
and COI genes based on sequence data from 236 different
mammals.
2. Materials and methods
Sequence data for the cyt b and COI genes from 236 mammals,
compromising 29 Orders, and a large number of individual human,
cow and dog sequences were obtained from the NCBI Genome
database (available http://www.ncbi.nlm.nih.gov/). Sequences
were from complete mitochondrial genome records and the cyt
b and COI genes were extracted.

1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2009.08.053
 S.S. Tobe et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 306–307
Fig. 1. Pie charts representing the proportion of homologous and variable sites
within the cyt b gene (A) and COI gene (B). The values are represented as a
percentage of base sites over the largest variant of the relevant gene (1149 and
1557 bp, for cyt b and COI, respectively).
Sequences were imported into MEGA 4.0 [8,9] and aligned
(other mammals separately from humans, cows and dogs).
Following alignment, sequences were transferred to Excel (Micro-
soft) for analysis. Once in Excel, sequences were analyzed to
determine homologous and variable positions.
307
Intra-species variation was assessed by performing the same
analysis using multiple members of the same species for human,
cow and dog samples.
3. Results and conclusions
The size of the cyt b and COI genes was found to be variable
between different species, but invariable within the same species.
Each gene showed a size variation to a maximum of 20 bp between
different species with cyt b ranging from 1130 to 1149 bp and COI
ranging from 1537 to 1557 bp.
Of the total 1149 base pairs (bp) of cyt b, 22.4% (257 bp) were
conserved in all samples (Fig. 1A). COI contained 680 sites out of
1557 which were conserved in all samples, or 43.7% of the total
gene (Fig. 1B). There were small sections that contained either a
greater number of variable or homologous bases, however the
variation was generally evenly spread over the entire genes.
Intra-species variation was assessed by analyzing a large
number of human, cow and dog samples. The cyt b gene in human,
cow and dog samples was found to be 82%, 97% and 98%
homologous, respectively. Homology in the COI gene was found
to be 88%, 97% and 98% for human, cow and dog, respectively.
This simple sequence analysis demonstrates that, for mamma-
lian samples, the use of the cytochrome b gene will offer greater
informative value in a smaller fragment. Intra-species variation is
similar in both genes for the same species indicating that one is not
better than another in regards to intra-species variation. This is
ideal when dealing with trace or degraded samples.
Funding
This project was funded by the Leverhulme Trust (Grant
number A20080076) awarded to AL.
Conflict of interest
None.
Acknowledgement
None.
References
[1] T.D. Kocher, et al., Dynamics of mitochondrial DNA evolution in animals: ampli-
fication and sequencing with conserved primers, Proceedings of the National
Academy of Sciences of the United States of America 86 (16) (1989) 6196–6200.
[2] D. Irwin, T. Kocher, A. Wilson, Evolution of the cytochrome b gene of mammals,
Journal of Molecular Evolution 32 (2) (1991) 128–144.
[3] H.-M. Hsieh, et al., Cytochrome b gene for species identification of the conservation
animals, Forensic Science International 122 (1) (2001) 7–18.
[4] P.D.N. Hebert, et al., Biological identifications through DNA barcodes, Proceedings
of the Royal Society B: Biological Sciences 270 (1512) (2003) 313–321.
[5] C. Moritz, C. Cicero, DNA barcoding: promise and pitfalls, PLoS Biology 2 (10) (2004)
e354.
[6] M. Vences, et al., Comparative performance of the 16S rRNA gene in DNA barcoding
of amphibians, Frontiers in Zoology 2 (5) (2005).
[7] F. Mayer, C. Dietz, A. Kiefer, Molecular species identification boosts bat diversity,
Frontiers in Zoology 4 (1) (2007) 4.
[8] S. Kumar, et al., MEGA: a biologist-centric software for evolutionary analysis of
DNA and protein sequences, Briefings in Bioinformatics 9 (4) (2008) 299–306.
[9] K. Tamura, et al., MEGA4: Molecular Evolutionary Genetics Analysis (MEGA)
software version 4.0, Molecular Biology and Evolution 24 (8) (2007) 1596–1599.