The antisense insertion of a canine short interspersed element (SINEC_Cf) in the pigmentation gene PMEL (or SILV) causes a coat pattern phenotype in dogs termed merle. Merle is a semi-dominant trait characterized by patches of full pigmentation on a diluted background. The oligo(dT) tract of the Merle retrotransposon is long and uninterrupted and is prone to dramatic truncation. Phenotypically wild-type individuals carrying shorter oligo(dT) lengths of the Merle allele have been previously described and termed cryptic merles. Two additional coat patterns, dilute merle (uniform, steely-grey coat) and harlequin merle (white background with black patches), also appear in breeds segregating the Merle allele. Sequencing of all PMEL exons in a dilute and a harlequin merle reveals that variation exists solely within the oligo(dT) tract of the SINEC_Cf insertion. In fragment analyses from 259 dogs heterozygous for Merle, we observed a spectrum of oligo(dT) lengths spanning 25 to 105 base pairs (bp), with ranges that correspond to the four varieties of the merle phenotype: cryptic (25–55 bp), dilute (66–74 bp), standard (78–86 bp), and harlequin (81–105 bp). Somatic contractions of the oligo(dT) were observed in 43% of standard and 51% of harlequin merle dogs. A small proportion (4.6%) of the study cohort inherited de novo contractions or expansions of the Merle allele that resulted in dilute or harlequin coat patterns, respectively. The phenotypic consequence of the Merle SINE insertion directly depends upon oligo(dT) length. In transcription, we propose that the use of an alternative splice site increases with oligo(dT) length, resulting in insufficient PMEL and a pigment dilution spectrum, from dark grey to complete hypopigmentation. We further propose that during replication, contractions and expansions increase in frequency with oligo(dT) length, causing coat variegation (somatic events in melanocytes) and the spontaneous appearance of varieties of the merle phenotype (germline events).

The genomic diversity of the domestic dog is an invaluable resource for advancing understanding of mammalian biology, evolutionary biology, morphologic variation, and behavior. There are approximately 350 recognized breeds in the world today, many established through hybridization and selection followed by intense breeding programs aimed at retaining or enhancing specific traits. As a result, many breeds suffer from an excess of particular diseases, one of many factors leading to the recent trend of “designer breed” development, i.e. crossing purebred dogs from existing breeds in the hope that offspring will be enriched for desired traits and characteristics of the parental breeds. We used a dense panel of 150,106 SNPs to analyze the population structure of the Australian labradoodle (ALBD), to understand how such breeds are developed. Haplotype and admixture analyses show that breeds other than the poodle (POOD) and Labrador retriever (LAB) contributed to ALBD formation, but that the breed is, at the genetic level, predominantly POOD, with all small and large varieties contributing to its construction. Allele frequency analysis reveals that the breed is enhanced for variants associated with a poodle-like coat, which is perceived by breeders to have an association with hypoallergenicity. We observed little enhancement for LAB-specific alleles. This study provides a blueprint for understanding how dog breeds are formed, highlighting the limited scope of desired traits in defining new breeds. Due to the selective breeding practices used to create modern breeds, dogs suffer from an unacceptable excess of disease, one of several reasons behind the recent trend of creating “designer breeds.” Such populations result from crosses between established breeds, with a goal of producing dogs that lack or have a reduced incidence of unfavorable disorders or phenotypes, yet retain desirable traits from parental breeds. One such example is the Australian labradoodle which was initiated from Labrador retriever and standard poodle crosses 31 years ago in order to produce dogs with a reduced tendency to elicit an allergic reaction but that retained the desirable characteristics of a service dog. In this study we have used nuclear DNA markers to determine the genetic history of the Australian labradoodle and develop an understanding of how breeds stabilize traits given particular breeding strategies. Our data shows that despite the major contributions from poodle and Labrador retriever, additional breeds contributed to the modern Australian labradoodle. Today’s Australian labradoodle is largely poodle with an excess of poodle alleles related to coat type. This study demonstrates that changes in very few genes can define a new breed and demonstrates how breeds can form in a small number of generations.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 (P_wald= 4.83x10^-9) and 19 (P_wald= 2.25x10^-7). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway genePIK3R6on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation ofTNFAIP6, which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.

The lack of an annotated reference sequence for the canine Y chromosome has limited evolutionary studies, as well as our understanding of the role of Y-linked sequences in phenotypes with a sex bias. In genome-wide association studies (GWASs), we observed spurious associations with autosomal SNPs when sex was unbalanced in case-control cohorts and hypothesized that a subset of SNPs mapped to autosomes are in fact sex-linked. Using the Illumina 230K CanineHD array in a GWAS for sex, we identified SNPs that amplify in both sexes but possess significant allele frequency differences between males and females. We found 48 SNPs mapping to 14 regions of eight autosomes and the X chromosome that are Y-linked, appearing heterozygous in males and monomorphic in females. Within these 14 regions are eight genes: three autosomal and five X-linked. We investigated the autosomal genes (MITF, PPP2CB, and WNK1) and determined that the SNPs are diverged nucleotides in retrocopies that have transposed to the Y chromosome. MITFY and WNK1Y are expressed and appeared recently in the Canidae lineage, whereas PPP2CBY represents a much older insertion with no evidence of expression in the dog. This work reveals novel canid Y chromosome sequences and provides evidence for gene transposition to the Y from autosomes and the X.

Significance: Through the study of a dental anomaly we identified a locus strongly associated with body size in the Shetland Sheepdog. Within this locus are variants in two genes: a substitution in FtsJ RNA 2′-O-Methyltransferase 3 ( FTSJ3 ) and a splice donor insertion in Growth Hormone 1 ( GH1 ). We demonstrated that the GH1 variant causes an abnormal splicing pattern that is also observed in dominant forms of human pituitary dwarfism. Interestingly, the FTSJ3 variant is estimated to have the greatest impact on height and weight and this gene has not been previously characterized in body size traits. Both derived alleles are found in high frequencies in very small “toy” breeds but are entirely absent from larger breeds.

Background: Histiocytic sarcoma (HS) is a highly aggressive malignancy characterized by the excessive proliferation of histiocytes in dogs and humans. A subset of dog breeds, including the Bernese Mountain Dog (BMD), show a remarkably high prevalence of HS. Previous work by us and others has identified somatic driver mutations of HS in the PTPN11 and KRAS genes that activate the MAPK pathway in about 60% of canine HS. However, no somatic driver mutations have been identified in the remaining 40%. Objectives: Our goals are to study HS in BMDs to gain insight into the molecular pathogenesis of the disease, and identify rational approaches to therapy. Methods: Here, we report our whole transcriptome analysis of 18 well-characterized BMD HS tumor tissues, as well as three HS cell lines. Results: Our analysis reveals the significant upregulation of molecular pathways involving the FOXM1, AURKB, PLK1, and E2F genes, in HS as well as hemophagocytic HS, providing new information regarding pathways that may be targeted with inhibitors. In addition, we document the expression of multiple checkpoint genes, suggesting the option of treatment with small-molecule inhibitors together with checkpoint inhibitors. Further, we show that the transcriptomes of three canine HS cell lines mirror those of canine patient tumors, further highlighting their potential use in drug discovery and efficacy studies. Finally, we demonstrate, for the first time, that aurora kinase inhibitors are effective in curtailing the growth of HS cells in vitro and show synergism with MAPK inhibition. Conclusions: This study provides the most detailed analysis of the canine HS transcriptome to date, highlighting key pathways in its pathogenesis and suggesting new avenues for both single and combination treatment strategies, which may be pertinent to the treatment of human HS.