An STR Forensic Typing System for Genetic Individualization of Domestic Cat (Felis catus) Samples

Abstract

A forensic genotyping panel of 11 tetranucleotide STR loci from the domestic cat was characterized and evaluated for genetic individualization of cat tissues. We first examined 49 candidate STR loci and their frequency assessment in domestic cat populations. The STR loci (3-4 base pair repeat motifs), mapped in the cat genome relative to 579 coding loci and 255 STR loci, are well distributed across the 18 feline autosomes. All loci exhibit Mendelian inheritance in a multi-generation pedigree. Eleven loci that were unlinked and were highly heterozygous in cat breeds were selected for a forensic panel. Heterozygosity values obtained for the independent loci, ranged from 0.60-0.82, while the average cat breed heterozygosity obtained for the 11 locus panel was 0.71 (range of 0.57-0.83). A small sample set of outbred domestic cats displayed a heterozygosity of 0.86 for the 11 locus panel. The power of discrimination of the panel is moderate to high in the cat breeds examined, with an average P m of 3.7E-06. The panel shows good potential for genetic individualization within outbred domestic cats with a P m of 5.31E-08. A multiplex protocol, designed for the co-amplification of the 11 loci and a gender-identifying locus, is species specific and robust, generating a product profile with as little as 0.125 nanograms of genomic DNA.

Using population genetic tools to develop a control strategy for feral cats (Felis catus) in Hawai'i

“…We generated complete genotypes for all feral cat samples using seven polymorphic short-tandem repeat (STR) microsatellite markers previously developed and optimised for multiplexing of domestic cats (Menotti-Raymond et al 2005) and included a gender-identifying sequence tagged site (STS) from the domestic cat Y-chromosome SRY gene. The markers were labelled with D2, D3 and D4 WellRED fluorescent dyes (Beckman Coulter, Fullerton, CA, USA) at the five-prime end of either the forward or reverse primers.…”

“…A single PCR (polymerase chain reaction) was performed for each sample in a 20.0-µL volume containing 1 × PCR Gold buffer (Applied Biosystems, Fullerton, CA, USA), with final reaction concentrations (adapted from Menotti- Raymond et al 2005) as follows: 1.5 mM MgCl 2 , 0.8 mM dNTPs, 1 unit Amplitaq gold DNA polymerase (Applied Biosystems), 0.16 mg mL -1 bovine serum albumin, 8 µL microsatellite primer mix (see final concentrations in Table 1), and~20 ng genomic DNA. PCRs were performed with a MJ Research PTC-200 DNA thermocycler, using conditions optimised by Menotti-Raymond et al (2005).…”

“…PCRs were performed with a MJ Research PTC-200 DNA thermocycler, using conditions optimised by Menotti-Raymond et al (2005). The eight-primer pair multiplexes were then visualised on a Beckman Coulter CEqn 8000 automated capillary sequencer, one lane for each DNA sample (Core Genetics Facility, University of Hawai'i at Hilo).…”

“…The eight-primer pair multiplexes were then visualised on a Beckman Coulter CEqn 8000 automated capillary sequencer, one lane for each DNA sample (Core Genetics Facility, University of Hawai'i at Hilo). Allele sizes were estimated using CEQN 8000 version 7.0 and then visually inspected, taking into consideration the expected allele size in base pairs for each of the eight loci and the original DNA clones from which the microsatellite loci were developed (Menotti- Raymond et al 2005).…”

“…A Bonferonni correction was used to adjust significance levels across multiple tests (Goudet 2001). All loci occurred on different chromosomes or different linkage groups on the same chromosome (Menotti- Raymond et al 2005) and were considered independent markers.…”