Comprehensive analysis of geographic and breed-purpose influences on genetic diversity and inherited disease risk in the Doberman dog breed.

BACKGROUND: Publicly available phenotype data and genotyping array data from two citizen science projects: "Doberman Health Surveys" and "The Doberman Diversity Project" were analyzed to explore relative homozygosity, diversity, and disorder risk according to geographical locale and breeding purpose in the Doberman. RESULTS: From the phenotypic data cohort, life expectancy of a Doberman at birth is 9.1 years. The leading causes of death were heart disease (accounting for 28% of deaths) and cancers (collectively accounting for 14% of deaths). By genotyping, the world Doberman population exists as four major cohorts (European exhibition-bred, Americas exhibition-bred, European work, Americas pet/informal). Considering the entire Doberman population, four genomic regions longer than 500 Kb are fixed in 90% or more of 3,226 dogs included in this study. The four fixed regions reside on two autosomal chromosomes: CFA3:0.8-2.3 Mb (1.55 Mb); CFA3: 57.9-59.8 Mb (1.8 Mb); CFA31:0-1.2 Mb (1.2 Mb); and CFA31:4.80-6.47 Mb (1.67 Mb). Using public variant call files including variants for eight Doberman pinschers, we observed 30 potentially functional alternate variants that were evolutionarily diverged relative to the wider sequenced dog population within the four strongly homozygous chromosomal regions. Effective population size (Ne) is a statistical measure of breed diversity at the time of sampling that approximates the number of unique individuals. The major identified sub-populations of Dobermans demonstrated Ne in the range 70-236. The mean level of inbreeding in the Doberman breed is 40% as calculated by the number of array variants in runs of homozygosity divided by the assayed genome size (excluding the X chromosome). The lowest observed level of inbreeding in the Dobermans assayed was 15% in animals that were first generation mixes of European and USA bred Dobermans. Array variant analysis shows that inter-crossing between European and USA-bred Dobermans has capacity to re-introduce variation at many loci that are strongly homozygous. CONCLUSIONS: We conclude that efforts to improve breed diversity first should focus on regions with the highest fixation levels, but managers must ensure that mutation loads are not worsened by increasing the frequencies of rarer haplotypes in the identified regions. The analysis of global data identified regions of strong fixation that might impact known disorder risks in the breed. Plausible gene candidates for future analysis of the genetic basis of cardiac disease and cancer were identified in the analysis.

Genetic Variants at the Nebulette Locus Are Associated with Myxomatous Mitral Valve Disease Severity in Cavalier King Charles Spaniels.

The most common cardiovascular disease in domestic dogs is myxomatous mitral valve disease (MMVD), accounting for 75% of all cardiac disease. An increase in age is generally associated with increased incidence of the disease, but Cavalier King Charles Spaniels (CKCS) exhibit an unusually high prevalence of early-onset MMVD, and thus, potentially greater cardiac morbidity and mortality compared to other breeds. Previous research has suggested that selected candidate risk alleles for MMVD are fixed in CKCSs, including six locations within the Nebulette (NEBL) gene on CFA2. The current study analysed genotypes of 180 Australian CKCSs at the identified risk loci. Of these, 178 were phenotyped for severity of disease by echocardiographic measurements of left atrium to aortic root ratio (LA:Ao) and weight normalised left ventricular end diastolic diameter (LVIDdN). Genotyping array markers correctly predicted the genotype at the risk-variant loci in the CKCS population, and the NEBL1, NEBL2 and NEBL3 variants were observed to be in perfect linkage disequilibrium in this cohort. The CKCS cohort included 6/178 dogs being heterozygous for the protective/wild-type alleles at the NEBL locus. The mean LA:Ao and LVIDdN scores of these dogs heterozygous at NEBL1-3 variants were significantly smaller, and with significantly lower variance compared to age-matched CKCSs that were homozygous for risk alleles. The lower cardiac measurements in the heterozygous dogs indicate a significantly reduced risk of severe MMVD disease. Our analysis suggests that despite relative fixation of the NEBL risk alleles, healthy reference alleles at NEBL1-3 exist in low frequency in the CKCS breed and can be used to reduce MMVD severity and mortality.

Cerebellar Abiotrophy in Australian Working Kelpies Is Associated with Two Major Risk Loci.

An autosomal recessive form of inherited cerebellar abiotrophy (CA) that is characterized by a degeneration of Purkinje and granule cells in the cerebellar cortex occurs in the Australian working kelpie dog breed. The clinical signs of CA include ataxia, head tremor, motor in-coordination, wide-based stance, and high-stepping gait. Investigation of clinical and pathological features indicated two closely related diseases with differences in age of onset. A genome-wide association study on 45 CA affected and 290 normal healthy Kelpies identified two significantly associated loci, one on CFA9 and a second on CFA20. Dogs homozygous for the risk haplotype on CFA20 (23 dogs) show clinical signs before ten weeks of age. Missense variants in the sixth exon of disruptor of telomeric silencing 1-like (DOT1Lp.R200Q) and in the only exon of Leucine Rich Repeat And Ig Domain Containing 3 (LINGO3p.R359C), both on CFA20, segregate with the associated risk marker which has incomplete penetrance (42%). Affected dogs homozygous for the risk haplotype on CFA9 have later onset ataxia. A missense variant in exon 5 of Vacuole Membrane Protein 1 (VMP1 p.P160Q) on CFA9 segregates as a fully penetrant Mendelian recessive with later-onset CA. Across mammals, the variety of causative loci so far identified as influencing cerebellar disorders reinforces the complexity of the pathways that contribute to cerebellar development and function, and to the pathophysiological mechanisms that may lead to cerebellar ataxia.

Genome-Wide Association Analysis for Chronic Superficial Keratitis in the Australian Racing Greyhound.

Chronic superficial keratitis (CSK) is a progressive inflammatory condition of the eye (cornea) that can cause discomfort and blindness. Differential disease risk across dog breeds strongly suggests that CSK has a genetic basis. In addition to genetic risk, the occurrence of CSK is exacerbated by exposure to ultraviolet light. Genome-wide association analysis considered 109 greyhounds, 70 with CSK and the remainder with normal phenotype at an age over four years. Three co-located variants on CFA18 near the 5' region of the Epidermal Growth Factor Receptor (EGFR) gene were associated with genome-wide significance after multiple-test correction (BICF2P579527, CFA18: 6,068,508, praw = 1.77 × 10-7, pgenome = 0.017; BICF2P1310662, CFA18: 6,077,388, praw = 4.09 × 10-7, pgenome = 0.040; BICF2P160719, CFA18: 6,087,347, praw = 4.09 × 10-7, pgenome = 0.040) (canFam4)). Of the top 10 associated markers, eight were co-located with the significantly associated markers on CFA18. The associated haplotype on CFA18 is protective for the CSK condition. EGFR is known to play a role in corneal healing, where it initiates differentiation and proliferation of epithelial cells that in turn signal the involvement of stromal keratocytes to commence apoptosis. Further validation of the putative functional variants is required prior to their use in genetic testing for breeding programs.

Valued personality traits in livestock herding Kelpies-Development and application of a livestock herding dog assessment form.

Livestock herding dogs contribute greatly to the rural economy of Australia. However, their selection currently lacks a cohesive or methodical approach. For example, there is no accessible tool for assessing Australian livestock herding dogs' suitability for work. The purpose of the current study was to devise a herding dog assessment form, the Herding Dog Assessment Form-Personality (HDAF-P), to facilitate collection of data on relevant behavioural phenotypes of large numbers of working Kelpies and to apply the HDAF-P to identify personality traits needed for herding dog performance. The focus was on creating a succinct form that was salient and accessible to livestock herding dog owners. Wherever practical, terms and methods from published personality questionnaires were integrated. Seventeen terms were included as behavioural descriptors in the HDAF-P which was then used by 95 owners to assess a sample of 228 of their working Kelpies. Owners were also asked to rate the overall ability of their dog(s). Of these dogs, 210 (all twelve months or older) were fully described and their data were used in the analysis. Thus, the study was designed to reveal which personality traits are most critical to the overall ability of the herding dogs and to undertake an exploratory analysis of the patterns of dog behaviour revealed by the HDAF-P in non-juvenile dogs. The traits that showed the strongest correlations (using Kendall's Tau correlation analysis) with overall ability were initiative (T = 0.41, p < 0.001), persistence (T = 0.36, p < 0.001), intelligence (T = 0.32, p < 0.001), confidence (T = 0.36, p < 0.001) and nervousness (T = -0.30, p < 0.001). An exploratory principal component analysis of trait scores revealed that 64.5% of the variance could be explained by four components that share several similarities with those reported by previous dog personality studies. These findings confirm that the HDAF-P has potential for the practical assessment of livestock herding dog personality and can elucidate traits that should be considered for prioritisation in training and breeding to optimise herding dog ability.

Forensic DNA phenotyping: Canis familiaris breed classification and skeletal phenotype prediction using functionally significant skeletal SNPs and indels.

This review highlights a novel application of breed identification and prediction of skeletal traits in forensic investigations using canine DNA evidence. Currently, genotyping methods used for canine breed classification involve the application of highly polymorphic short tandem repeats in addition to larger commercially available SNP arrays. Both applications face technical challenges. An additional approach to breed identification could be through genotyping SNPs and indels that characterise the array of skeletal differences displayed across domestic dog populations. Research has shown that a small number of genetic variants of large effect drive differences in skeletal phenotypes among domestic dog breeds. This feature makes functionally significant canine skeletal variants a cost-effective target for forensic investigators to classify individuals according to their breed. Further analysis of these skeletal variants would enable the prediction of external appearance. To date, functionally significant genes with genetic variants associated with differences in size, bulk, skull shape, ear shape, limb length, digit type, and tail morphology have been uncovered. Recommendations of a cost-effective genotyping method that can be readily designed and applied by forensic investigators have been given. Further advances to improve the field of canine skeletal forensic DNA phenotyping include the refinement of phenotyping methods, further biological validation of the skeletal genetic variants and establishing a publicly available database for storage of allele frequencies of the skeletal genetic variants in the wider domestic dog population.

Exploiting genomic synteny in Felidae: cross-species genome alignments and SNV discovery can aid conservation management.

BACKGROUND: While recent advances in genomics has enabled vast improvements in the quantification of genome-wide diversity and the identification of adaptive and deleterious alleles in model species, wildlife and non-model species have largely not reaped the same benefits. This has been attributed to the resources and infrastructure required to develop essential genomic datasets such as reference genomes. In the absence of a high-quality reference genome, cross-species alignments can provide reliable, cost-effective methods for single nucleotide variant (SNV) discovery. Here, we demonstrated the utility of cross-species genome alignment methods in gaining insights into population structure and functional genomic features in cheetah (Acinonyx jubatas), snow leopard (Panthera uncia) and Sumatran tiger (Panthera tigris sumatrae), relative to the domestic cat (Felis catus). RESULTS: Alignment of big cats to the domestic cat reference assembly yielded nearly complete sequence coverage of the reference genome. From this, 38,839,061 variants in cheetah, 15,504,143 in snow leopard and 13,414,953 in Sumatran tiger were discovered and annotated. This method was able to delineate population structure but limited in its ability to adequately detect rare variants. Enrichment analysis of fixed and species-specific SNVs revealed insights into adaptive traits, evolutionary history and the pathogenesis of heritable diseases. CONCLUSIONS: The high degree of synteny among felid genomes enabled the successful application of the domestic cat reference in high-quality SNV detection. The datasets presented here provide a useful resource for future studies into population dynamics, evolutionary history and genetic and disease management of big cats. This cross-species method of variant discovery provides genomic context for identifying annotated gene regions essential to understanding adaptive and deleterious variants that can improve conservation outcomes.

Inbreeding levels in an open-registry pedigreed dog breed: The Australian working kelpie.

The depletion in genetic diversity of closed-pedigree dog breeds can be a contentious topic and can lead to calls for open-registry. However, strong evidence in support of proposed open-registry solutions is lacking, with the reproductive isolation of these breeds unlikely to be the sole cause of elevated inbreeding levels. Human-induced limitations, such as popular sire effects, are unlikely to be confined to closed-registry breeds and conceivably play an important role in maintaining genetic diversity within all breeds. Consequently, the aim of the current study was to explore inbreeding levels in an open-registry breed and determine the impact open-registry has on genetic diversity. Complete pedigree records on all Australian working kelpies (AWKs) were provided by the Working Kelpie Council with the cleaned pedigree consisting of 86,671 individuals with a median pedigree depth of 6.6 generations. The average inbreeding coefficient in the AWK population was 0.049 with an increase in inbreeding coefficient of 0.0016/year. This demonstrates that opening a breed registry can have a beneficial impact on the level of inbreeding within a population over the longer-term. However, allowing for a generation length of 5.1 years yielded an effective population size of 61 for AWKs and demonstrated a pattern consistent with closed-registry dog populations of comparable size.

Mapping the genetic basis of diabetes mellitus in the Australian Burmese cat (Felis catus).

Diabetes mellitus, a common endocrinopathy affecting domestic cats, shares many clinical and pathologic features with type 2 diabetes in humans. In Australia and Europe, diabetes mellitus is almost four times more common among Burmese cats than in other breeds. As a genetically isolated population, the diabetic Australian Burmese cat provides a spontaneous genetic model for studying diabetes mellitus in humans. Studying complex diseases in pedigreed breeds facilitates tighter control of confounding factors including population stratification, allelic frequencies and environmental heterogeneity. We used the feline SNV array and whole genome sequence data to undertake a genome wide-association study and runs of homozygosity analysis, of a case-control cohort of Australian and European Burmese cats. Our results identified diabetes-associated haplotypes across chromosomes A3, B1 and E1 and selective sweeps across the Burmese breed on chromosomes B1, B3, D1 and D4. The locus on chromosome B1, common to both analyses, revealed coding and splice region variants in candidate genes, ANK1, EPHX2 and LOX2, implicated in diabetes mellitus and lipid dysregulation. Mapping this condition in Burmese cats has revealed a polygenic spectrum, implicating loci linked to pancreatic beta cell dysfunction, lipid dysregulation and insulin resistance in the pathogenesis of diabetes mellitus in the Burmese cat.

A large deletion on CFA28 omitting ACSL5 gene is associated with intestinal lipid malabsorption in the Australian Kelpie dog breed.

Inborn errors of metabolism are genetic conditions that can disrupt intermediary metabolic pathways and cause defective absorption and metabolism of dietary nutrients. In an Australian Kelpie breeding population, 17 puppies presented with intestinal lipid malabsorption. Juvenile dogs exhibited stunted postnatal growth, steatorrhea, abdominal distension and a wiry coat. Using genome-wide association analysis, an associated locus on CFA28 (Praw = 2.87E-06) was discovered and validated in a closely related population (Praw = 1.75E-45). A 103.3 kb deletion NC_006610.3CFA28:g.23380074_23483377del, containing genes Acyl-CoA Synthetase Long Chain Family Member 5 (ACSL5) and Zinc Finger DHHC-Type Containing 6 (ZDHHC6), was characterised using whole transcriptomic data. Whole transcriptomic sequencing revealed no expression of ACSL5 and disrupted splicing of ZDHHC6 in jejunal tissue of affected Kelpies. The ACSL5 gene plays a key role in long chain fatty acid absorption, a phenotype similar to that of our affected Kelpies has been observed in a knockout mouse model. A PCR-based diagnostic test was developed and confirmed fully penetrant autosomal recessive mode of inheritance. We conclude the structural variant causing a deletion of the ACSL5 gene is the most likely cause for intestinal lipid malabsorption in the Australian Kelpie.

Characterization of A Homozygous Deletion of Steroid Hormone Biosynthesis Genes in Horse Chromosome 29 as A Risk Factor for Disorders of Sex Development and Reproduction.

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7-29.9 Mb. The region contains AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in AKR1C genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8-9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders.

Association between coat colour and the behaviour of Australian Labrador retrievers.

BACKGROUND: Making assumptions regarding temperament and intelligence based on the physical appearance of dogs can be a conscious or unconscious human act. Labrador retrievers with chocolate-coloured coats are anecdotally considered to be less trainable and more hyperactive and aggressive than their black or yellow peers. To test these assertions, we analysed the owner-reported behavioural traits of Labradors in relation to both their observable coat colour, and their TYRP1 and MC1R genotypes. RESULTS: We used the results of an owner-based questionnaire to determine scores for 21 behavioural traits and test whether these scores varied with coat colour (n = 225). Familiar dog aggression was the only trait that was found to vary significantly with coat colour (P = 0.013). Yellow Labradors had a higher score than chocolate Labradors, even when corrected for multiple testing (P = 0.021).We repeated the analyses for a subset of 63 Labradors with available genotyping data for the genes (MC1R and TYRP1) that are known to determine the primary coat colours in Labradors. Familiar dog aggression scores varied with both the observed coat colour and MC1R genotype. Dogs homozygous for MC1R recessive allele (with yellow coat colour) scored higher for familiar dog aggression than either black or chocolate Labradors. However, no association maintained significance when incorporating Bonferroni correction. Dog trainability scores decreased additively as the number of recessive brown alleles for TYRP1 increased. This allelic association was independent of the observable coat colour. Dogs homozygous for the brown allele were considered less trainable than dogs with no brown alleles (P = 0.030). CONCLUSIONS: Our results do not support that chocolate-coloured Labradors are more hyperactive or aggressive than either black or yellow Labradors. Trainability scores varied with TYRP1 genotype but not the observable coat colour. Further validation is required.

Genomic Characterization of External Morphology Traits in Kelpies Does Not Support Common Ancestry with the Australian Dingo.

The Kelpie is a breed developed in Australia for use as a livestock herding dog. It has been proposed that the development of the breed included gene flow from the Australian Dingo (Canis dingo), a canid species present on the Australian continent for around 4000 years. The Kelpie breed is split between working and conformation types that have readily recognizable differences in external morphology. We characterize known gene variants relating to external morphology in sequenced representatives of both Kelpie types (Australian Kelpie-conformation; Australian Working Kelpie-herding) and compare the variants present with those in sequenced Australian Dingoes, including 25 canids with locus-constrained data and one with a whole genome sequence. Variants assessed include identified coat color and ear morphology variants. We describe a new variant site in the transcribed region of methionine sulfoxide reductase 3 that may relate to ear phenotype. None of the morphology variants analyzed offer support for co-ancestry of the Kelpie breed with the Australian Dingo.

Work-type influences perceived livestock herding success in Australian Working Kelpies.

BACKGROUND: Working dog handlers and breeders have very different behavioural requirements in the animals that they employ for managing livestock. The Australian Working Kelpie breed may be used in several working contexts, notably yards, paddocks and a combination of both. The working context influences the skillsets required and gives rise to three corresponding work-types: Yard, Paddock and Utility Kelpies. In particular, dogs used for working stock in the confines of yards and trucks interact with stock more forcefully than those mustering in larger areas (paddocks) where they can herd stock effectively from a greater distance. This article explores owner assessments of dog working quality and assessment of genomic similarity by multidimensional scaling, to ask whether it is sufficient for breeders to aim for a multipurpose breeding objective, or whether breeding only specialist lines maximises user satisfaction for yard and paddock work. RESULTS: Reported owner perceptions of 298 dogs assessed with the Livestock Herding Dog assessment tool showed that dog handlers across all working types were very happy with their dogs' level of general skills.Compared with both Yard and Utility Kelpies, Paddock Kelpies had significantly lower trait scores for force (pressure applied by the dog to move livestock), willingness to back the stock (run along a sheep's dorsum) and bite (frequency of using the mouth to grab or bite the livestock). Meanwhile, compared with both Paddock and Utility Kelpies, the Yard Kelpies had significantly higher scores for hyperactivity and excitability (both with and without stock) and impulsiveness without stock. As one would predict for all-rounders, Utility Kelpies had intermediate scores for all behaviours and working traits. CONCLUSIONS: Specialist characteristics were displayed by dogs in the Yard Kelpie and Paddock Kelpie groups. In particular, Yard Kelpies demonstrate higher excitability, willingness to back the stock, and a higher tendency to bark and bite the stock. Conversely, Paddock Kelpies rarely display these characteristics. Utility Kelpies, as the name suggests, are intermediate between the other two groups and display the characteristics of both. Genetic analysis suggests that the Yard, Utility and Paddock Kelpies are not distinguishable at a DNA level. In conclusion, at this time there is no suggestion of a breed split in the Australian Working Kelpie generated by selection for work type. A common breeding objective should enable dogs to be produced that fulfil all potential working requirements. This reinforces the importance of breeder skill in recognising the phenotypic potential of pups in order to place them in appropriate working contexts.

Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.