Mitochondrial DNA sequencing of cat hair: an informative forensic tool

Abstract

Approximately 81.7 million cats are in 37.5 million U.S. households. Shed fur can be criminal evidence because of transfer to victims, suspects, and/or their belongings. To improve cat hairs as forensic evidence, the mtDNA control region from single hairs, with and without root tags, was sequenced. A dataset of a 402-bp control region segment from 174 random-bred cats representing four U.S. geographic areas was generated to determine the informativeness of the mtDNA region. Thirty-two mtDNA mitotypes were observed ranging in frequencies from 0.6-27%. Four common types occurred in all populations. Low heteroplasmy, 1.7%, was determined. Unique mitotypes were found in 18 individuals, 10.3% of the population studied. The calculated discrimination power implied that 8.3 of 10 randomly selected individuals can be excluded by this region. The genetic characteristics of the region and the generated dataset support the use of this cat mtDNA region in forensic applications.

FIG. 1

Domestic cat mtDNA control region (modified from Lopez et al., 1996). (a) Physical map of coding genes within the cat cytoplasmic mtDNA. Genes on the inner circle are transcribed from the light (L) strand. Locations of the tRNA genes (shaded boxes) conform to the canonical placental mammalian arrangement and are presented as their standard single letter abbreviations. The following additional abbreviations were used: HSP, putative heavy-strand promoter; OHR, origin of heavy-strand replication; OLR, origin of light-strand replication. (b) The domestic cat CR extends from nucleotide 16,315 to nucleotide 865, producing a 1,559 region. There is a 335-bp overlap of the CR sequence with Numt, which begins at bp 529 within the RS3 and extends to nucleotide 8,454 that includes ~80% of the COII gene. Two distinct repetitive motifs, RS2 and RS3, at opposite ends the CR contribute to the relatively large size of the cat’s mtDNA as compared to other carnivores. These sites are subject to high heteroplasmy. Placement of PCR primers for the three CR studies are noted with arrows.

FIG. 2

Sylvester reference sequence for domestic cat mtDNA CR. Presented is the majority rule consensus sequence generated from 1315 mtDNA CR sequences from domestic cats. The nucleotides presented in bold identify the 37 transition and transversion sites identified. Nucleotides adjacent to an insertion/deletion are underlined. Numbering is in reference to the PCR generated product/first mtDNA genomic sequence generated for the cat (.).

FIG. 3

Minimal Spanning Network of mtDNA CR Haplotypes for All Cat Populations. Specific populations are represented by colors: California (solid black), Hawaii (open squares), New York (stipled), and Texas (checkered squares). Unfilled wedges in H01 and H03 represent control sample mitotypes. Bold designators (H##) indicate haplotypes. Theoretical intermediary haplotypes are identified by diamond nodes. Samples without identifiers are unique. The composite “Sylvester” reference sequence is noted. Numbers on the branches indicate the positions and amount of mutations needed to derive connecting mitotypes.

FIG. 4

Minimal Spanning Network of mtDNA CR Haplotypes for Specific Cat Populations. a. California, b. Hawaii, c. New York, d. Texas. Black wedges represent control sample haplotypes. Bold designators (H##) indicate haplotypes. Theoretical mitotypes are designated solid diamonds. Nodes without identifiers are unique. The composite “Sylvester” reference sequence is noted. Numbers on the branches indicate the positions and amount of mutations needed to derive the connecting mitotype.