DNA markers for forensic identification of non-human biological traces Wesselink, M.

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1 UvA-DARE (Digital Academic Repository) DNA markers for forensic identification of non-human biological traces Wesselink, M. Link to publication Citation for published version (APA): Wesselink, M. (). DNA markers for forensic identification of non-human biological traces General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: or a letter to: Library of the University of Amsterdam, Secretariat, Singel, WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam ( Download date: Jan

2 Chapter Forensic utility of the feline mitochondrial control region A Dutch perspective M. Wesselink, L. Bergwerff, D. Hoogmoed, A.D. Kloosterman and I. Kuiper Forensic Science International: Genetics () - Abstract Different portions of the feline mitochondrial DNA control region (CR) were evaluated for their informative value in forensic investigations. The bp region located between RS and RS described most extensively in the past is not efficient for distinguishing between the majority of Dutch cats, illustrated by a random match probability (RMP) of %. Typing of the whole region between RS and RS, and additional typing of the portion of the feline CR decreases the RMP to %, increasing the applicability of such analyses for forensic investigations. The haplotype distribution in Dutch random bred cats (N = ) differs greatly from the distributions reported for other countries, with a single haplotype NL-A present in % of the population. The three investigated breeds showed haplotype distributions differing from each other and the random bred cats with haplotype NL-A accounting for %, % and % of Maine Coon, Norwegian forest cats and Siamese & Oriental cats. These results indicate the necessity of validating haplotype frequencies within continents and regions prior to reporting the value a mtdna match. In cases where known purebred cats are involved, further investigation of the breed may be valuable. Supplementary data associated with this article can be found in the online version

3 Chapter. Introduction In the Netherlands, a country with almost million human inhabitants, the number of cats is estimated to fluctuate around million [,]. As in other parts of the world, approximately % of these domestic cats are random bred, also referred to as European shorthair or nonbreed, the remaining % consisting of fancy breed cats [,]. In the densely inhabited parts of the Netherlands, some cats have an indoor life (mainly fancy breed cats), others pass time outdoor (mainly random bred cats). Few cats live independently of humans, due to human control of feral cat populations [,]. Due to this lifestyle and the continuous, year round shedding of hairs, cat hairs are easily spread to the belongings of people living in proximity of cats. When these people become involved in a crime, whether as a victim, witness or perpetrator, cat hairs can be transferred. Like hairs from other species, in this manner cat hairs can be silent witnesses of a crime, when for example they are transferred from a suspect s clothes to tape used to restrain a victim. If nuclear STR markers can be amplified from such trace evidence, the link between the trace evidence (a hair) and the donor (the cat) can be extremely informative []. In our experience, as also pointed out by others [,], amplifying nuclear DNA (ndna) markers from animal hairs encountered in case work is generally unsuccessful. The amplification of mitochondrial DNA (mtdna) from shed hairs has significantly higher success rates, mainly due to the high copy number and greater stability of the mtdna molecule and the distribution of mtdna in hairs []. This makes mtdna the DNA molecule of choice for forensic investigation of cat hairs, despite the fact that it is less informative than ndna due to its maternal inheritance and lack of recombination [,]. To use the mtdna from hairs as trace evidence, a reliable and robust technique is necessary with which a maximum of information can be obtained from such small traces containing only a limited amount of mtdna. The mtdna control region (CR), also referred to as displacement loop, D-loop and hypervariable regions (HV), has proven to be the most informative part of the mtdna in many species including humans [], dogs [] and cats [,]. In humans and dogs specific portions of the CR have been recognized that have diverse characteristics and therefore are of different value for forensic purposes. The extent to which the applicability of these regions has been investigated varies. The tandem repeat structures present in the feline CR (RS and RS) have been shown to be too variable for forensic applications, as the number of repeats may vary within an individual, often prohibiting complete sequencing of both strands of DNA []. Due to the presence of a numt and the possibility of amplifying ndna instead of mtdna [], the information content of the portion of the feline CR has only been investigated briefly []. The remaining two portions of the feline CR, the portion located before RS (also referred to as HVI and HVa) and the middle portion located between RS and RS (also referred to as HVII and

4 Forensic utility of the feline mitochondrial control region A Dutch perspective HVb) have both been described as forensically informative regions, but their informative value has not yet been compared. One of the factors in determining the value of a portion of the mtdna CR may be the availability of relevant reference samples for which this region has been typed []. Geographical origin, random bred vs fancy breed cats and human factors all influence whether publicly available data can be used. Several studies have focused on specific parts of the mtdna in specific breeds, random bred cats or wild cats in different regions. Portions of the mtdna of cats from islands (Japan and the United Kingdom) have been described [,], and from several mainland European countries (Italy, Spain and Poland) [,], however as different regions of the mtdna were used the data from these studies cannot be compared. Analysis of large number of cats from different regions of the United States resulted in comparable haplotype distributions, but differed from a relatively small sampling of non-us samples [] and analyses of non-us cats was advised. To investigate whether cat breeds could be distinguished by their mtdna, several individuals ( ) of different breeds mainly from the US were typed, however no breed specific haplotypes were identified []. This fits the observation that the development of cat breeds is a relatively recent process with most breeds originating within the last years and the fact that crossbreeding of cats from different breeds is permitted within breed standards as some are defined by coat pattern, hair length or color mutations alone. The genetic relationship and admixture between several breeds has been clearly shown by autosomal DNA studies [ ]. To be able to apply feline mtdna in forensic case work in The Netherlands a better understanding of random bred cats and fancy breed cats in The Netherlands and North West Europe is essential. Therefore we evaluated the value of the different portions of the feline mitochondrial control region for nucleotide diversity and random match probability within random bred cats from the Netherlands. We additionally explored substructuring of the domestic cat population in the Netherlands to establish a framework for interpretation of mtdna matches in Dutch casework.. Material and methods.. Sample collection A random sampling of Dutch random bred cats was obtained by collecting buccal swabs of cats owned by (relatives of) personnel of the Netherlands Forensic Institute living throughout the Netherlands. In cases where swabs of multiple littermates or parents and offspring had been collected, only one sample of each known maternal group was included in this study. This random sampling resulted in the collection of random bred cats (RBC, %), mixed breed cats, British Shorthairs, Maine Coons, Persians, Siamese, Bengal, Birman, and Siberian Forest cat, the majority collected in the most densely inhabited part of the country (distribution of RBC samples in Fig. ).

5 Chapter.. Laboratory procedures DNA was extracted from buccal swabs using the DNeasy Blood and Tissue kit (Qiagen GmbH, Germany) following the manufacturer s protocol. Total DNA concentration was estimated through spectrophotometry with a NanoDrop ND- (NanoDrop Technologies, DE, USA). A portion of the feline mtdna CR was amplified using forward primer FCB-Z [] and reverse primer JHmtR [] (Fig. ). PCR reactions were performed in μl containing x PCR Buffer (containing. mm MgCl),. mm MgCl,. unit of HotStarTaq. mm each dntp (all Qiagen), pmol of each primer and. ng of template DNA. PCRs were performed on MyCycler (BioRad Laboratories) or GeneAmp (Applied Fig.. Distribution of housholds, cats and RBC samples. Map generated with LocalFocus. Biosystems) thermocyclers using the following cycling parameters: min at C; cycles of C for s, C for s, and C for min; min at C. PCR success was determined by gel-electrophoresis followed by ethidium bromide staining and UV detection. Positive and negative controls were used throughout. ExoSAP-IT (Affymetrix) was used following the manufacturer s protocol for removal of unincorporated primers an dntps. μl of each purified PCR product was sequenced with the PCR primers and JHmtF [] in a total of μl, containing x BigDye sequencing buffer,.x BigDye Terminator v. Ready Reaction Mix (Life Technologies) and pmol primer. Cycling parameters were min at C; cylces of C for s, C for s and C for min. Residual sequencing components were removed using BigDye XTerminator Purification Kit (Life Technologies) following the manufacturer s protocol. Sequencing products were separated on an AB XL genetic analyzer, containing cm capillaries using POP- TM polymer (all Life Technologies)... Sequence analysis Sequences were evaluated using Geneious.. []. Forward and reverse sequences were assembled into contigs (containing the RS region) and basecalling of the non-rs region was checked and edited manually. Sequences containing ambiguities and sequence assemblies producing ambiguities were resequenced. Positions remaining heteroplasmic were denoted according to IUPAC nomenclature. After assembly, consensus sequences were aligned and trimmed to remove non-control region DNA and the RS repeat using Geneious. Nucleotide positions (NP) - and - (corresponding to Genbank U and NC_ []) were retained.

6 Forensic utility of the feline mitochondrial control region A Dutch perspective Variable nucleotide positions were identified in comparison with the first published feline mitochondrial DNA sequence []. The nucleotide positions of the published Sylvester reference sequence (SRS) [,] is included to facilitate comparisons with studies considering NP -, but was not used otherwise. Fig.. Domestic cat control region with nucleotide positions (NP) / as described by Lopez et al. []. RS consists of multiple repeats of appr. bp; RS consists of multiple repeats of appr. bp. The Numt begins at NP and extends to NP. Sequenced portions of the control region used in this study and in [ ], [] and [] are indicated, as are the positions of amplification primers FCB-Z [] and JHmtR []... Data analysis JmodelTest.. [,] was used to select the models best describing the complete bp region. The HKY model [] with invariable sites which assumes differences in base frequencies, and differences between the rates of transitions and transversions described the data most precisely. This model was used for construction of maximum likelihood trees in MEGA. [] (all sites, all haplotypes, bootstrap replicates). Random match probability (RMP) was calculated as (- p i ) where p is the frequency of the observed haplotype and discrimination capacity is -RMP. The unbiased haplotype diversity was calculated as N (- p ) []. A phylogeographic network of haplotypes was constructed N i with the median joining algorithm implemented in Network. (Fluxus Technology Ldt) [].. Results.. Characteristics of the different regions Amplicons of. to. kb were obtained from all buccal samples. From the majority of samples, fragments with varying lengths were obtained as visualized after gel-electrophoresis. Sequencing of the different fragments was not hampered by their presence. Sequences of on average bp were obtained from randomly selected cats (RSC). The different regions of the feline CR were analyzed using this randomly sampled cat data to evaluate the forensic utility of these regions. The four regions of interest were: the bp preceding RS (NP -); the bp corresponding to SRS; the bp between RS and RS (NP -

7 Chapter ); and the combination of the bp before RS and bp between RS and RS (Table ). The lowest discrimination capacity of approximately % was observed in the region (NP -), followed by % in the bp SRS region. The discrimination capacity of NP - exceeded the other regions (%), however the highest discrimination capacity was observed when the 'region preceding RS and the region between RS and RS were combined (%). The increase in informative value when comparing the regions before and after RS and these regions combined, is also demonstrated by the number of haplotypes, number of singletons and gene diversity (Table ). Based on these data, the combined region of bp was used for further analyses. Table. Comparison of segments of feline mtdna CR based on randomly sampled cats. bp before RS bp [,,] bp (RS-RS) Total bp - Based on RSC # haplotypes # singletons unbiased gene diversity (SD). ±.. ±.. ±.. ±. # matches/ comparisons random match probabiliy.... discrimination capacity Analysis of haplogroups The bp sequence of the combined regions (preceding RS and between RS and RS) were aligned for random bred cats (RBC), Maine Coon cats (MC), Norwegian Forest (NF) cats and Siamese and Oriental Shorthair cats with the first published feline mtdna sequence U as is common practice for human and canine mitochondrial DNA studies. This resulted in the recognition of different haplotypes. Through phylogenetic analysis of these haplotypes, six haplogroups (NL-A, NL-B, NL-C, NL-D, NL-E and NL-F) were recognized (Network diagram in Fig., Maximum Likelihood tree in supplementary data). Most of the haplotypes differed from the three most frequently occurring haplotypes by only one (%) or two (%) nucleotide substitutions. Fig.. Network diagram of all mtdna CR sequences without heteroplasmic positions encountered in this study. U [] is shown as a reference. Circles are scaled to frequencies for the different breed groups: RBC (white), MC (black), NF (dark gray) and S O (light grey). All differences between haplotypes are indicated.

8 Forensic utility of the feline mitochondrial control region A Dutch perspective haplotypes derived from individuals contained a heteroplasmic position. One haplotype (NL- A) was found to contain a bp deletion. Additional typing of a maternally related individual confirmed this deletion. All haplotypes were numbered and nucleotide positions polymorphic when compared to U are displayed in Table. The nucleotide position numbering of SRS is shown to facilitate comparison with other studies. Table. Haplotype defining polymorphic nucleotide positions.. [] U G C A G A Δ C A A C C T C C T G G C C C A C C T C T A A G T T C T T A G T C C A G T C G A T - T T T T T C G G NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C. A.. G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T... A. C C... C.. T A C... C... C A. T A A NL-A C.. C Δ... G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T. C T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T... G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C... C.. C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C.. T A C... C... C A. T A A NL-A C. T C.... G T T. T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... T.. T T.. T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T. T T G.. A. C C... C.. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A. T. A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C T. T A C... C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C.. T A C. A. C... C A. T A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A.. A A NL-A C.. C.... G T.. T.. T T.. T G.. A. C C... C.. T A C... C... C A. Y A A NL-D C.. C.... G T.. T.. T T.. T... A. C.. A... C C.... T T A C.. G C T A. C A. T A A NL-D C.. C Δ... G T.. T.. T T.. T... A. C.. A... C C.... T T A C.. G C.. A C A. T A A NL-B C.. C.. A. G T... A C T T A. C G G A..... G. C.. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T.... T. A. C G G A..... G. C.. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A. C G G A.. T.. G. C.. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A T C G G A..... G. C.. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A. C G G A..... G. C.. T A.. A. C. A.. A C T A A NL-B C.. C.. A. G T... A C T T A. C G G A..... G. C T. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A. C G G A..... G. C.. T A C... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A. C G G A C.. T A.... C. A.. A C T. A NL-B C.. C.. A. G T... A C T T A. C G G A..... G. C.. T A.... C. A.. A C.. A NL-B C.. C.. A. G T... A C T T C G G A. C... G. C.. T A.... C. A.. A C.. A NL-B C T. C.. A. G T... A C T T C G G A. C... G. C.. T A.... C. A.. A C.. A NL-B C.. C.. A. G T... A C T T C G G A. C..... C.. T A.... C. A.. A... A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C... G.... T A.. A C T. A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C Y.. G.... T A.. A C T. A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C.. C G.... T A.. A C T. A NL-C C.. C.... G T..... T T T. T.. T A. C G G.. C... G.... T C A.. A C T. A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C... G. C.. T A.. A C T. A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C... G A... T A.. A C T. A NL-C C.. C.... G T..... T T.. T.. T A. C G G.. C... G.... T A.. A. T. A NL-E C.. C.... G T..... T T.. T... A. C G G.. C.. C.... T T A. T A. C T A.. A... A NL-F C.. C T T A... A SRS [] Dots indicate positions identical to the reference sequence U []. Base calls (IUPAS nomenclature) show the substitutions at specified positions (Y=C/T), Δ indicates a deletion, indicates a bp deletion from NP - encountered in a single haplotype. Grey nucleotide positions are the same in all encountered haplotypes, but differ from the reference U. Nucleotide positions of SRS [] are indicated for comparative purposes only..

9 Chapter.. Comparison of breed groups The distribution of the haplotypes within the random bred cats, MC, NF and S O is shown in Table. A large variation in haplotype distribution, haplogroup distribution and genetic diversity was observed between the four groups of cats. The vast majority of random bred cats (%) has a haplotype in haplogroup A, % have a haplotype within haplogroup B, whereas haplogroups C, D, E and F are only represented by a total of % of the random bred cats. This is in contrast with the three breed groups, where in MCs only % is placed in haplogroup A, % in haplogroup B, % in haplogroup E, and all others (%) in haplogroup C. In NF haplogroups A, C and D each account for approximately % of haplotypes, with the remaining % belonging to haplogroup B. Siamese and Oriental Shorthairs exhibited only haplotypes belonging to haplogroups A (%) and B (%). To examine a possible effect of mainly sampling RBC from the western part of The Netherlands, frequency distributions from the western, middle and eastern part of The Netherlands were compared. No pronounced differences were observed (data not shown). Of the haplotypes, were present in more than % of individuals within at least one breed. The most frequently encountered haplotype, NL-A is carried by more than half of all random bred cats. This haplotype is encountered less frequently in Maine Coon, Norwegian Forest and Siamese & Oriental Shorthairs (%, % and %, respectively). Haplotype NL-B was encountered in % of Maine Coons and % of Siamese & Oriental Shorthairs, while it was only seen in % of random bred cats and % of Norwegian Forest cats. Haplotypes NL- C and NL-E were observed in % and % of Maine Coons, respectively, but not in the other cats breeds or random bred cats. Haplotype NL-D was found in % of Norwegian Forest cats and in a single random bred cat. Of the haplotypes, were encountered only in a single individual.. Discussion In this study sequencible amplicons were obtained from all tested buccal DNA extracts. Although the presence of the RS repeat in the center of the amplicon caused amplicons to increase in length up to. kb, this did not have a negative effect on PCR or sequence success of the tested samples. Amplification of this large fragment from single hair DNA extracts as encountered in casework, can be hampered when only a limited amount of possibly degraded DNA is present (data not shown). In such cases the use of multiple smaller amplicons is needed to obtain both regions of the CR. Ideally a single amplicon is preferred to minimize costs and the risk of laboratory sample mix up. However the increase in information when both the CR region prior to and the region directly after RS are used outweighs these disadvantages. Although the portion of feline mtdna CR located between RS and RS has been most studied most extensively and this region is more informative than the region before RS, it is not sufficient to distinguish between the majority of the Dutch cats. To increase the information that can be retrieved from a single cat hair, especially when a sample is typed as haplogroup NL-A, it is advisable to include the region before RS even if this implies

10 Forensic utility of the feline mitochondrial control region A Dutch perspective Table. Haplotype distribution within random bred cats and three breed groups. Random bred cats Maine Coon Norwegian Forest cats Siamese & Oriental total # frequency # frequency # frequency # frequency # NL-A.... NL-A.. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-A. NL-D.. NL-D. NL-B.... NL-B. NL-B.. NL-B. NL-B. NL-B. NL-B. NL-B. NL-B. NL-B.. NL-B. NL-B. NL-C... NL-C. NL-C. NL-C. NL-C. NL-C. NL-C. NL-E. NL-F. N # haplotypes # singletons % singleton samples..... unbiased gene diversity (SD). ±.. ±.. ±.. ±. # matches/# comparisons / / / / random match probabiliy.... discrimination capacity.... amplification and sequencing of additional amplicons. By addition of this region, the frequency of the largest halpogroup (%) is reduced to %. As this one group is still very large, the added value of even more regions of the mtdna should be considered. Whole genome sequencing of the mtdna could be used to determine regions or SNPs of interest, but other regions previously used to distinguish felines should not be forgotten. Cytb and NADH are logical candidate regions, as is the region of the CR especially when applied to hairs with no ndna present. The typing of this region in such hairs should not pose any difficulties, although typing reference buccal swabs might could well cause difficulties due to the numt present in the ndna. The collection of not only reference buccal swabs but also reference hair samples could circumvent this problem.

11 Chapter.. Geographical comparisons The bp SRS region of the mtdna CR of the random bred cats was used to enable comparison of our dataset with previously published haplotype frequencies from the United States (n = ) [], European mainland (Germany n = and Italy n = ) [] and the United Kingdom (n = ) []. As shown in Fig. A, the frequencies of the major haplotypes encountered in this study differ greatly from the frequencies of these haplotypes in other studies. Some resemblance can be seen between the data from this study and a small sampling of European mainland cats published previously, but discrepancy between the observed frequency of haplogroup A and the frequency of unique haplotypes is pronounced. Although no statistical analyses were performed, the greatest difference between the frequencies observed in this study and in the US and UK studies is in the frequencies of haplogroups A and C. When calculated for this bp region, the unbiased gene diversity of the Dutch RBC is only. ±., which is far lower than values calculated for European mainland (. ±., n = ) and American RBC (. ±., n = ) []. The haplotype distribution of the Dutch RBCs was also compared to domestic cats from Japan (Tsushima Islands, n = ) [] utilizing only the portion of CR sequences (Fig. B). This resulted in comparable results, with haplogroup NL-A highly overrepresented in the Dutch population and NL-B and NL-C underrepresented. Several new haplotypes were detected in the Dutch population and a type found in % of the Japanese samples was not observed in The Netherlands. When calculated for this bp region, the unbiased gene diversity of the Dutch RBC is only. ±., which is substantially lower than the. ±. calculated for the Japanese cats (n = ) []. Fig.. Comparison of haplotype distribution of RBCs with previous feline mtdna CR studies [,]and []. A: bp region between RS and RS. B: sequence before RS. Multiple biological and reproduction factors related to human and cat population density, sterilization and breeding practices that may well differ between countries or regions, as well as the number of wild living/stray cats may contribute to the observed differences between sample sets. As cat movement between The Netherlands and the United Kingdom is thought to be less frequent than between The Netherlands and other mainland North-West European

12 Forensic utility of the feline mitochondrial control region A Dutch perspective countries, additional typing of cats from the eastern part of The Netherlands, Belgium and Germany is desirable. This may indicate if the frequencies described in this study are typical for a North-West European distribution, or whether the distribution in The Netherlands differs from that in its neighboring countries... Breed comparisons In line with previous studies [,], no direct relationship between breed and haplogroup or haplotype was observed. This fits the description of domestication of cats, and the relatively recent origin of fancy cat breeds. However the haplogroups (and haplotype) distribution between the studied cat breeds differed notably. This has implications for forensic casework if the same mtdna profile is found in a crime scene sample and in a reference cat and the reference cat is known to be a fancy breed cat. As only three breed groups were tested, no conclusions can be drawn about fancy cat breeds in general. As many different cat breeds are recognized and some are (intentionally) cross bred with others, obtaining a reasonable number of samples of these breeds seems unfeasible. However if such a situation occurs during a case investigation, it may be valuable to type additional unrelated cats of the same breed to allow better evaluation of the evidence.. Conclusion Our results show that mtdna haplotype NL-A is highly overrepresented in random bred cats from The Netherlands. As the vast majority of Dutch cats are random bred, chances of encountering a hair with haplotype NL-A in casework are high. When examination of a reference animal (or other trace evidence) shows that animal also has haplotype NL-A, the evidential value of such a match will generally be deemed small as the random match probability is high (~%). However if a casework hair and reference cat both have a non-nl- A haplotype, the evidential value can rise dramatically as the probability of encountering an uncommon mtdna haplotype in a Dutch cat is approximately %. The impact of cat hairs in forensic investigations may increase even further, when hairs from multiple unrelated cats are transferred during a crime. Although the portion of feline mtdna CR located between RS and RS has been studied most extensively and this region is more informative than the region before RS, it is not sufficient to distinguish between the majority of the Dutch cats. To increase the information that can be retrieved from a cat hairs, especially when a sample is typed as haplogroup NL-A, it is advisable to additionally type the region before RS. Comparison of the frequency distributions of Dutch cat mtdna haplotypes with distributions from other studies indicate that it is undesirable to utilize frequency distributions from other continents or regions without validating these frequencies for a particular region.

13 Chapter Acknowledgements We appreciate all private cat owners and catteries who contributed cat DNA samples and pedigrees for this study. We also thank Jon Wetton, University of Leicester, UK for permission to reproduce the UK mtdna CR frequency distributions. Arnoud Kal NFI, and two anonymous reviewers are thanked for valuable comments on the manuscript. References [] HAS Kennistransfer. Feiten & cijfers gezelschapsdierensector. Hogeschool HAS Den Bosch;. Dutch. [] FEDIAF facts & figures [Internet]. The European Pet Food Industry Federation; Brussels, Belgium. [cited October ] Available from: [] What Kind of Cat Do You Want? [Internet]. The Cat Fanciers' Association; United States of America. [cited May ]. Available from: [] D.R. Lammertsma, R. Janssen, J. van der Hout, H.A.H. Jansman, D.R. Lammertsma, R. Janssen, J. van der Hout, H.A.H. Jansman, Huiskatten in natuurgebieden; Kan TNR hybridisatie met de Wilde kat voorkomen? Alterra, Wageningen University, Dutch,. [] Ministry of Economic Affairs. Uitvoering motie afschieten van katten.. March. Dutch. [] M.A. Menotti-Raymond, V.A. David, S.J. O Brien, Pet cat hair implicates murder suspect, Nature (), doi: [] J.L. Halverson, C. Basten, Forensic DNA identification of animal-derived trace evidence: tools for linking victims and suspects, Croat. Med. J. (). [] L.A. Lyons, R.A. Grahn, T.J. Kun, L.R. Netzel, E.E. Wictum, J.L. Halverson, Acceptance of domestic cat mitochondrial DNA in a criminal proceeding, Forensic Sci. Int. Genet. (), doi: [] C.A. Linch, Degeneration of nuclei and mitochondria in human hairs, J. Forensic Sci. (), doi: [] B. Budowle, M.W. Allard, M.R. Wilson, R. Chakraborty, Forensics and mitochondrial DNA: applications, debates and foundations, Annu. Rev. Genom. Hum. Genet. (). [] R.E. Giles, H. Blanc, H.M. Cann, D.C. Wallace, Maternal inheritance of human mitochondrial DNA, Proc. Natl. Acad. Sci. U. S. A. (). [] S. Horai, K. Hayasaka, Intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial DNA, Am. J. Hum. Genet. () (). [] P. Savolainen, B. Rosén, A. Holmberg, T. Leitner, M. Uhlén, J. Lundeberg, Sequence analysis of domestic dog mitochondrial DNA for forensic use, J. Forensic Sci. (). [] J.L. Halverson, L.A. Lyons, Forensic DNA identification of feline hairs: casework and a mitochondrial database, Proc. Am. Acad. Forensic Sci. () B. [] F. Fridez, S. Rochat, R. Coquoz, Individual identification of cats and dogs using mitochondrial DNA tandem repeats? Sci. Justice (), doi: (),-.

14 Forensic utility of the feline mitochondrial control region A Dutch perspective [] J.V. Lopez, S. Cevario, S.J. O Brien, Complete nucleotide sequences of the domestic cat (Felis catus) mitochondrial genome and a transposed mtdna tandem repeat (Numt) in the nuclear genome, Genomics () (), doi: [] W. Branicki, A. Olszanska, M. Konopinski, Sequence variation in the control region of mitochondrial DNA within a population sample of domestic cats Felis catus linnaeus implications for domestic and wild cats differentiation, Prob. Forensic Sci. (). [] T. Tamada, N. Kurose, R. Masuda, Genetic diversity in domestic cats Felis catus of the Tsushima Islands, based on mitochondrial DNA cytochrome b and control region nucleotide sequences, Zool. Sci. () (), doi: [] J.H. Wetton, B. Ottolini, G. Matharu Lall, M.A. Jobling, A Forensic Database of Cat Mitochondrial DNA Variants, DNA in Forensics, Brussels,, pp. P. [] E. Randi, M. Pierpaoli, M. Beaumont, B. Ragni, A. Sforzi, Genetic identification of wild and domestic cats (Felis silvestris) and their hybrids using Bayesian clustering methods, Mol. Biol. Evol. () (). [] R.A. Grahn, J.D. Kurushima, N.C. Billings, J.C. Grahn, J.L. Halverson, E. Hammer, et al., Feline non-repetitive mitochondrial DNA control region database for forensic evidence, Forensic Sci. Int. Genet. () (), doi: [] M.J. Lipinski, L. Froenicke, K.C. Baysac, N.C. Billings, C.M. Leutenegger, A.M. Levy, et al., The ascent of cat breeds: genetic evaluations of breeds and worldwide random-bred populations, Genomics () (), doi: [] M. Menotti-Raymond, V.A. David, S.M. Pflueger, K. Lindblad-Toh, C.M. Wade, S.J. O Brien, W.E. Johnson, Patterns of molecular genetic variation among cat breeds, Genomics () (), doi: [] J.D. Kurushima, M.J. Lipinski, B. Gandolfi, L. Froenicke, J.C. Grahn, R.A. Grahn, L.A. Lyons, Variation of cats under domestication: genetic assignment of domestic cats to breeds and worldwide random-bred populations, Anim. Genet. () (), doi: [] H. Alhaddad, R. Khan, R.A. Grahn, B. Gandolfi, J.C. Mullikin, S.A. Cole, et al., Extent of linkage disequilibrium in the domestic cat, Felis silvestris catus, and its breeds, PLoS One () () e, doi: [] C.R. Tarditi, R.A. Grahn, J.J. Evans, J.D. Kurushima, L.A. Lyons, Mitochondrial DNA. sequencing of cat hair: an informative forensic tool, J. Forensic. Sci. (s) () S S, oi: -...x. [] A.J. Drummond, B. Ashton, M. Cheung, J. Heled, M. Kearse, R. Moir, et al., Geneious v., Biomatters Ltd, Auckland, New Zealand,. [] D. Darriba, G.L. Taboada, R. Doallo, D. Posada, jmodeltest : more models, new heuristics and parallel computing, Nat. Methods () (), doi: [] S. Guindon, O. Gascuel, A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood, Syst. Biol. (), doi: [] M. Hasegawa, H. Kishino, T. Yano, Dating of the human-ape splitting by a molecular clock of mitochondrial DNA, J. Mol. Evol. () (), doi: [] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, MEGA: Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance, and maximum

15 Chapter parsimony methods, Mol. Biol. Evol. (), doi: [] D.A. Jones, Blood samples: probability of discrimination, J. Forensic Sci. Soc. () (), doi: [] H.-J. Bandelt, P. Forster, A. Röhl, Median-joining networks for inferring intraspecific phylogenies, Mol. Biol. Evol. ().