Kathryn R. Robertson

A comprehensive linkage map of the dog genome.

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dogs unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map.

An ADAMTSL2 Founder Mutation Causes Musladin-Lueke Syndrome, a Heritable Disorder of Beagle Dogs, Featuring Stiff Skin and Joint Contractures

Background Musladin-Lueke Syndrome (MLS) is a hereditary disorder affecting Beagle dogs that manifests with extensive fibrosis of the skin and joints. In this respect, it resembles human stiff skin syndrome and the Tight skin mouse, each of which is caused by gene defects affecting fibrillin-1, a major component of tissue microfibrils. The objective of this work was to determine the genetic basis of MLS and the molecular consequence of the identified mutation. Methodology and Principal Findings We mapped the locus for MLS by genome-wide association to a 3.05 Mb haplotype on canine chromosome 9 (CFA9 (50.11–54.26; praw <10−7)), which was homozygous and identical-by-descent among all affected dogs, consistent with recessive inheritance of a founder mutation. Sequence analysis of a candidate gene at this locus, ADAMTSL2, which is responsible for the human TGFβ dysregulation syndrome, Geleophysic Dysplasia (GD), uncovered a mutation in exon 7 (c.660C>T; p.R221C) perfectly associated with MLS (p-value = 10−12). Murine ADAMTSL2 containing the p.R221C mutation formed anomalous disulfide-bonded dimers when transiently expressed in COS-1, HEK293F and CHO cells, and was present in the medium of these cells at lower levels than wild-type ADAMTSL2 expressed in parallel. Conclusions/Significance The genetic basis of MLS is a founder mutation in ADAMTSL2, previously shown to interact with latent TGF-β binding protein, which binds fibrillin-1. The molecular effect of the founder mutation on ADAMTSL2 is formation of disulfide-bonded dimers. Although caused by a distinct mutation, and having a milder phenotype than human GD, MLS nevertheless offers a new animal model for study of GD, and for prospective insights on mechanisms and pathways of skin fibrosis and joint contractures.

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.

PCR multiplexed microsatellite panels to expedite canine genetic disease linkage analysis.

ABSTRACT Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25 centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.

Variation in Genes Related to Cochlear Biology Is Strongly Associated with Adult-Onset Deafness in Border Collies

Domestic dogs can suffer from hearing losses that can have profound impacts on working ability and quality of life. We have identified a type of adult-onset hearing loss in Border Collies that appears to have a genetic cause, with an earlier age of onset (3–5 years) than typically expected for aging dogs (8–10 years). Studying this complex trait within pure breeds of dog may greatly increase our ability to identify genomic regions associated with risk of hearing impairment in dogs and in humans. We performed a genome-wide association study (GWAS) to detect loci underlying adult-onset deafness in a sample of 20 affected and 28 control Border Collies. We identified a region on canine chromosome 6 that demonstrates extended support for association surrounding SNP Chr6.25819273 (p-value = 1.09×10−13). To further localize disease-associated variants, targeted next-generation sequencing (NGS) of one affected and two unaffected dogs was performed. Through additional validation based on targeted genotyping of additional cases (n = 23 total) and controls (n = 101 total) and an independent replication cohort of 16 cases and 265 controls, we identified variants in USP31 that were strongly associated with adult-onset deafness in Border Collies, suggesting the involvement of the NF-κB pathway. We found additional support for involvement of RBBP6, which is critical for cochlear development. These findings highlight the utility of GWAS–guided fine-mapping of genetic loci using targeted NGS to study hereditary disorders of the domestic dog that may be analogous to human disorders.

Marker panels for genealogy-based mapping, breed demographics, and inference-of-ancestry in the dog.

Short tandem repeat polymorphisms (STRPs) are robust and informative markers for a range of genetic applications. STRPs are advantageous in experimental designs that derive power from sampling many individuals rather than many loci (e.g., pedigree-based studies, fine-scale mapping, and conservation genetics). STRPs have proven useful for vetting samples prior to costly high-density SNP analysis. Here we present validated STRPs (n = 1,012) spanning the canine genome (2.1 +/−1.4 Mb; 2.1 +/−2.1 cM). Standardized design, pre-multiplexing, M13-based dye-labeling, and selection for loci amenable to semi-automated allele-scoring minimize cost and facilitate efficient genotyping. The markers are leveraged from the canine linkage map, and thus are backed by genetic data useful for parametric multipoint analysis and assessment of empiric coverage. We demonstrate several applications with different marker subsets. The complete set provides a genome scan for linkage at ∼5 cM resolution. A subset of the markers measures molecular diversity between domestic and wild canid populations. Another subset reflects ancestry within breeds, uncovering hidden stratification and flagging genetic outliers prior to SNP genotyping. Thus, the markers described here add flexibility and cost effectiveness to several genetic applications in the dog that complement genome-wide SNP genotyping studies. Supplemental material is available for this article. Go to the publishers online edition of Animal Biotechnology.

Semen preparation with the Sperm Select system versus a washing technique*†‡*Sperm Select; Select Medical Systems, Williston, Vermont.†Supported by a grant from Select Medical Systems, Williston, Vermont.‡Presented at the 41st Annual Meeting of the Pacific Coast Fertility Society, Indian Wells, California, April 14 to 18, 1993.

OBJECTIVE To compare sperm migration through sodium hyaluronate with simple washing as methods for preparing sperm for IUI. DESIGN Ten normal semen specimens were prospectively collected and samples were prepared by simple washing and by migration into sodium hyaluronate using the Sperm Select System (Select Medical Systems, Williston, VT). The semen and each treatment group were evaluated for sperm concentration, percent motile, viability, acrosomal status, longevity, and computer-aided semen analysis (CASA) parameters. SETTING University reproductive endocrinology facility. RESULTS The recovery of motile sperm was significantly higher for the washing method (mean +/- SEM 75% +/- 7%) than for the hyaluronate method (10% +/- 1%). The number of motile sperm recovered by migration into hyaluronate was independent of the percentage of motile sperm in the semen specimen and positively correlated with sperm concentration. The hyaluronate method produced greater percentages of motile, viable, and morphologically normal sperm, with lower proportions of premature acrosome reactions, higher sperm velocity, and greater linearity. CONCLUSIONS The Sperm Select System method of sperm separation provides a highly uniform specimen with improved sperm quality. However, the recovery of motile sperm is considerably lower than for simple washing methods.