Research Article| Volume 14, P86-95, January 2015

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Coding region SNP analysis to enhance dog mtDNA discrimination power in forensic casework

  • Sophie Verscheure
    Corresponding author at: National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, B-1120 Brussels, Belgium. Tel.: +32 22400541.
    National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, B-1120 Brussels, Belgium

    University of Antwerp (Evolutionary Ecology Group), Groenenborgerlaan 171, B-2020 Antwerp, Belgium
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  • Thierry Backeljau
    University of Antwerp (Evolutionary Ecology Group), Groenenborgerlaan 171, B-2020 Antwerp, Belgium

    Royal Belgian Institute of Natural Sciences (OD “Taxonomy and Phylogeny” and JEMU), Vautierstraat 29, B-1000 Brussels, Belgium
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  • Stijn Desmyter
    National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, B-1120 Brussels, Belgium
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Published:September 15, 2014DOI:


      • The control region of 132 newly sampled dogs was sequenced and added to a Belgian population study of 214 dogs.
      • Three SNP assays were developed targeting 26 mtDNA coding region sites that resolve the three most frequent CR haplotypes.
      • The set of 26 SNPs divide 139 dogs with the three most frequent CR haplotypes into 25 mtGenome clusters.
      • Using the SNP assays with CR sequencing increased the exclusion probability of dog mtDNA analysis from 92.9% to 97.0%.


      The high population frequencies of three control region haplotypes contribute to the low discrimination power of the dog mtDNA control region. It also diminishes the evidential power of a match with one of these haplotypes in forensic casework. A mitochondrial genome study of 214 Belgian dogs suggested 26 polymorphic coding region sites that successfully resolved dogs with the three most frequent control region haplotypes.
      In this study, three SNP assays were developed to determine the identity of the 26 informative sites. The control region of 132 newly sampled dogs was sequenced and added to the study of 214 dogs. The assays were applied to 58 dogs of the haplotypes of interest, which confirmed their suitability for enhancing dog mtDNA discrimination power. In the Belgian population study of 346 dogs, the set of 26 sites divided the dogs into 25 clusters of mtGenome sequences with substantially lower population frequency estimates than their control region sequences. In case of a match with one of the three control region haplotypes, using these three SNP assays in conjunction with control region sequencing would augment the exclusion probability of dog mtDNA analysis from 92.9% to 97.0%.


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        • Holland M.M.
        • Parsons T.J.
        Mitochondrial DNA sequence analysis – validation and use for forensic casework.
        Forensic Sci. Rev. 1999; 11: 21-50
        • Kim K.S.
        • Lee S.E.
        • Jeong H.W.
        • Ha J.H.
        The complete nucleotide sequence of the domestic dog (Canis familiaris) mitochondrial genome.
        Mol. Phylogenet. Evol. 1998; 10: 210-220
        • Savolainen P.
        • Lundeberg J.
        Forensic evidence based on mtDNA from dog and wolf hairs.
        J. Forensic Sci. 1999; 44: 77-81
        • Schneider P.M.
        • Seo Y.
        • Rittner C.
        Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident.
        Int. J. Legal Med. 1999; 112: 315-316
        • Branicki W.
        • Kupiec T.
        • Pawłowski R.
        Analysis of dog mitochondrial DNA for forensic identification purposes.
        Probl. Forensic Sci. (Z Zagadnien Nauk Sadowych). 2002; 50: 91-98
        • Aaspõllu A.
        • Kelve M.
        The first criminal case in Estonia with dog's DNA data admitted as evidence.
        Int. Congr. Ser. 2003; 1239: 847-851
        • Halverson J.L.
        • Basten C.
        Forensic DNA identification of animal-derived trace evidence: tools for linking victims and suspects.
        Croat. Med. J. 2005; 46: 598-605
        • Scharnhorst G.
        • Kanthaswamy S.
        An assessment of scientific and technical aspects of closed investigations of canine forensics DNA – case series from the University of California, Davis, USA.
        Croat. Med. J. 2011; 52: 280-292
        • Desmyter S.
        • Gijsbers L.
        Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing.
        Forensic Sci. Int. Genet. 2012; 6: 113-120
        • Verscheure S.
        • Backeljau T.
        • Desmyter S.
        Dog mitochondrial genome sequencing to enhance dog mtDNA discrimination power in forensic casework.
        Forensic Sci. Int. Genet. 2014; 12: 60-68
        • Savolainen P.
        • Zhang Y.P.
        • Luo J.
        • Lundeberg J.
        • Leitner T.
        Genetic evidence for an East Asian origin of domestic dogs.
        Science. 2002; 298: 1610-1613
        • Verscheure S.
        • Backeljau T.
        • Desmyter S.
        Reviewing population studies for forensic purposes: dog mitochondrial DNA.
        Zookeys. 2013; 365: 381-411
        • Webb K.M.
        • Allard M.W.
        Mitochondrial genome DNA analysis of the domestic dog: identifying informative SNPs outside of the control region.
        J. Forensic Sci. 2009; 54: 275-288
        • Imes D.L.
        • Wictum E.J.
        • Allard M.W.
        • Sacks B.N.
        Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes.
        Forensic Sci. Int. Genet. 2012; 6: 630-639
        • Angleby H.
        • Oskarsson M.
        • Pang J.
        • Zhang Y.P.
        • Leitner T.
        • Braham C.
        • et al.
        Forensic informativity of ∼3000 bp of coding sequence of domestic dog mtDNA.
        J. Forensic Sci. 2014;
        • Koressaar T.
        • Remm M.
        Enhancements and modifications of primer design program Primer3.
        Bioinformatics. 2007; 23: 1289-1291
        • Untergasser A.
        • Cutcutache I.
        • Koressaar T.
        • Ye J.
        • Faircloth B.C.
        • Remm M.
        • et al.
        Primer3 – new capabilities and interfaces.
        Nucleic Acids Res. 2012; 40: e115
        • Zuker M.
        Mfold web server for nucleic acid folding and hybridization prediction.
        Nucleic Acids Res. 2003; 31: 3406-3415
        • Vallone P.M.
        • Butler J.M.
        AutoDimer: a screening tool for primer-dimer and hairpin structures.
        Biotechniques. 2004; 37: 226-231
        • Kaplinski L.
        • Andreson R.
        • Puurand T.
        • Remm M.
        MultiPLX: automatic grouping and evaluation of PCR primers.
        Bioinformatics. 2005; 21: 1701-1702
        • Altschul S.F.
        • Madden T.L.
        • Schaffer A.A.
        • Zhang J.
        • Zhang Z.
        • Miller W.
        • et al.
        Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
        Nucleic Acids Res. 1997; 25: 3389-3402
        • Larkin M.A.
        • Blackshields G.
        • Brown N.P.
        • Chenna R.
        • McGettigan P.A.
        • McWilliam H.
        • et al.
        Clustal W and Clustal X version 2.0.
        Bioinformatics. 2007; 23: 2947-2948
        • Tamura K.
        • Peterson D.
        • Peterson N.
        • Stecher G.
        • Nei M.
        • Kumar S.
        MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
        Mol. Biol. Evol. 2011; 28: 2731-2739
        • Pereira L.
        • van Asch B.
        • Amorim A.
        Standardisation of nomenclature for dog mtDNA D-loop: a prerequisite for launching a Canis familiaris database.
        Forensic Sci. Int. 2004; 141: 99-108
        • Savolainen P.
        • Leitner T.
        • Wilton A.N.
        • Matisoo-Smith E.
        • Lundeberg J.
        A detailed picture of the origin of the Australian dingo, obtained from the study of mitochondrial DNA.
        Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12387-12390
        • Angleby H.
        • Savolainen P.
        Forensic informativity of domestic dog mtDNA control region sequences.
        Forensic Sci. Int. 2005; 154: 99-110
        • Pang J.F.
        • Klütsch C.
        • Zou X.J.
        • Zhang A.B.
        • Luo L.Y.
        • Angleby H.
        • et al.
        mtDNA data indicate a single origin for dogs south of Yangtze River, less than 16,300 years ago, from numerous wolves.
        Mol. Biol. Evol. 2009; 26: 2849-2864
        • Ardalan A.
        • Klütsch C.F.
        • Zhang A.B.
        • Erdogan M.
        • Uhlén M.
        • Houshmand M.
        • et al.
        Comprehensive study of mtDNA among Southwest Asian dogs contradicts independent domestication of wolf, but implies dog-wolf hybridization.
        Ecol. Evol. 2011; 1: 373-385
        • Klütsch C.F.
        • Seppälä E.H.
        • Fall T.
        • Uhlén M.
        • Hedhammar Å.
        • Lohi H.
        • et al.
        Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia.
        Anim. Genet. 2011; 42: 100-103
        • Oskarsson M.C.
        • Klütsch C.F.
        • Boonyaprakob U.
        • Wilton A.
        • Tanabe Y.
        • Savolainen P.
        Mitochondrial DNA data indicate an introduction through Mainland Southeast Asia for Australian dingoes and Polynesian domestic dogs.
        Proc. R. Soc. B. 2012; 279: 967-974
        • Excoffier L.
        • Lischer H.E.
        Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows.
        Mol. Ecol. Resour. 2010; 10: 564-567
        • Vallone P.M.
        • Just R.S.
        • Coble M.D.
        • Butler J.M.
        • Parsons T.J.
        A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome.
        Int. J. Legal Med. 2004; 118: 147-157
        • Sanchez J.J.
        • Endicott P.
        Developing multiplexed SNP assays with special reference to degraded DNA templates.
        Nat. Protoc. 2006; 1: 1370-1378
        • Podini D.
        • Vallone P.M.
        SNP genotyping using multiplex single base primer extension assays.
        Methods Mol. Biol. 2009; 578: 379-391
        • Børsting C.
        • Rockenbauer E.
        • Morling N.
        Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard.
        Forensic Sci. Int. Genet. 2009; 4: 34-42
        • Wilson M.R.
        • DiZinno J.A.
        • Polanskey D.
        • Replogle J.
        • Budowle B.
        Validation of mitochondrial DNA sequencing for forensic casework analysis.
        Int. J. Legal Med. 1995; 108: 68-74
        • Parr R.L.
        • Maki J.
        • Reguly B.
        • Dakubo G.D.
        • Aguirre A.
        • Wittock R.
        • et al.
        The pseudo-mitochondrial genome influences mistakes in heteroplasmy interpretation.
        BMC Genomics. 2006; 7: 185
        • Goios A.
        • Prieto L.
        • Amorim A.
        • Pereira L.
        Specificity of mtDNA-directed PCR-influence of NUclear MTDNA insertion (NUMT) contamination in routine samples and techniques.
        Int. J. Legal Med. 2008; 122: 341-345