Standardization of a SNP panel for parentage verification and identification in the domestic cat (Felis silvestris catus).

The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats.

Targeted genotyping by sequencing: a new way to genome profile the cat.

Targeted GBS is a recent approach for obtaining an effective characterization for hundreds to thousands of markers. The high throughput of next-generation sequencing technologies, moreover, allows sample multiplexing. The aims of this study were to (i) define a panel of single nucleotide polymorphisms (SNPs) in the cat, (ii) use GBS for profiling 16 cats, and (iii) evaluate the performance with respect to the inference using standard approaches at different coverage thresholds, thereby providing useful information for designing similar experiments. Probes for sequencing 230 variants were designed based on the Felis_catus_8.0. 8.0 genome. The regions comprised anonymous and non-anonymous SNPs. Sixteen cat samples were analysed, some of which had already been genotyped in a large group of loci and one having been whole-genome sequenced in the 99_Lives Cat Genome Sequencing Project. The accuracy of the method was assessed by comparing the GBS results with the genotypes already available. Overall, GBS achieved good performance, with 92-96% correct assignments, depending on the coverage threshold used to define the set of trustable genotypes. Analyses confirmed that (i) the reliability of the inference of each genotype depends on the coverage at that locus and (ii) the fraction of target loci whose genotype can be inferred correctly is a function of the total coverage. GBS proves to be a valid alternative to other methods. Data suggested a depth of less than 11× is required for greater than 95% accuracy. However, sequencing depth must be adapted to the total size of the targets to ensure proper genotype inference.

Informative enzyme/probe combinations for the multilocus DNA fingerprinting of marsupials.

DNA fingerprinting has become an invaluable tool in the study of population genetics, paternity success, and individual identification; however, the species specificity of some methods has made the wide-range screening of many different species very time-consuming. In this study we describe the development and application of reliable and informative DNA fingerprinting techniques in a range of marsupial species using three different restriction enzyme and two oligonucleotide probe combinations. Six species from four marsupial families, the koala (Phascolarctidae), tammar wallaby (Macropodidae), southern hairy-nosed wombat (Vombatidae), kowari, and dusky and brown marsupial mice (Dasyuridae) were examined. Restriction enzymes HinfI AluI and HaeIII were used in combination with the digoxygenin (DIG)-labelled oligonucleotide probes (CAC)5 and (GGAT)4. The combinations of HinfI/(GGAT)4, AluI/ (CAC)5 and AluI/(GGAT)4 were the most informative, providing highly resolved bands, low background, and the lowest band sharing between individuals. The genetic diversity evident within the different species showed a clear relationship between the level of band sharing and population size. The greatest levels of band sharing were found in the kowaris (80%), which were part of a long-term captive colony originating from a few founders, and the lowest levels of band sharing were found in the marsupial mice (30-35%) and tammar wallaby (45%), which were caught from large outbred wild populations.

DNA fingerprinting to determine paternity in laboratory rat sperm competition experiments.

Prior to this study a significant amount of research had been undertaken in the field of sperm competition in mammals. However, males of different strains have been required in each of these studies to enable paternity assignment through gene expression, which has consequently resulted in problems with differential fertilising capacity being encountered. In this study paternity assignment of progeny from sperm competition experiments with Sprague Dawley rats was achieved by multilocus DNA fingerprinting using band locus matching of individual specific banding patterns between progeny and parents. Trials with 4 restriction enzymes and 5 digoxygenin labelled probes (4 oligonucleotide and 1 cloned) achieved the highest levels of DNA fingerprint heterozygosity using AluI(CAC)5 and HinfI(CAC)5 combinations; however, paternity could not be determined in all offspring, due to a higher than expected degree of inbreeding within the rat population used in this study. This was demonstrated in subsequent comparisons of genetic diversity of three laboratory rat breeding populations from two different animal breeding facilities. Data from the rat mating study showed that, under conditions of direct sperm competition, second males given access to a mated oestrus female either 0.5 or 6.0 h after the first mating consistently required less time than the first to ejaculate: 7.6 min vs. 19.5 min (0.5 h delay ); 7.8 min vs. 19.5 min (6.0 h delay). A second males siring advantage was identified using DNA fingerprinting in both delay groups for those offspring on which paternity could be determined: 0.5 h delay, 1st = 39%, 2nd = 61%; 6 h delay, 1st = 34%, 2nd = 66%.