bioRxiv preprint doi: https://doi.org/10.1101/654343; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 True Colors: commercially-acquired morphological genotypes reveal hidden allele variation among 3 dog breeds, informing both trait ancestry and breed potential 4 5 (Using commercial genotype panels to inform dog breed trait variation) 6 7 8 Dayna L Dreger1¶, Blair N Hooser1¶, Angela M Hughes2, Balasubramanian Ganesan2, Jonas Donner3, 9 Heidi Anderson3, Lauren Holtvoigt2, Kari J Ekenstedt1* 10 11 1Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West 12 Lafayette, IN, USA 13 2Wisdom Health, Vancouver, WA, USA 14 3Wisdom Health, Helsinki, Finland 15 16 17 18 19 20 21 *Corresponding author email: [email protected] (KJE) 22 ¶These authors contributed equally to this work and are to be considered joint first authors. 23 24 25 26 bioRxiv preprint doi: https://doi.org/10.1101/654343; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 27 Abstract 28 Direct-to-consumer canine genetic testing is becoming increasingly popular among dog owners. 29 The data collected therein provides intriguing insight into the current status of morphological variation 30 present within purebred populations. Mars WISDOM PANELTM data from 11,790 anonymized dogs, 31 representing 212 breeds and 4 wild canine species, were evaluated at genes associated with 7 coat color 32 traits and 5 physical characteristics. Frequencies for all tested alleles at these 12 genes were determined 33 by breed and by phylogenetic grouping. A sub-set of the data, consisting of 30 breeds, was divided into 34 separate same-breed populations based on country of collection, body size, coat variation, or lineages 35 selected for working or conformation traits. Significantly different (p ≤ 0.00167) allele frequencies were 36 observed between populations for at least one of the tested genes in 26 of the 30 breeds. Next, standard 37 breed descriptions from major American and international registries were used to determine colors and 38 tail lengths (e.g. genetic bobtail) accepted within each breed. Alleles capable of producing traits 39 incongruous with breed descriptions were observed in 143 breeds, such that random mating within breeds 40 has probabilities of between 4.9e-7 and 0.25 of creating undesirable phenotypes. Finally, the presence of 41 rare alleles within breeds, such as those for the recessive black coloration and natural bobtail, was 42 combined with previously published identity-by-decent haplotype sharing levels to propose pathways by 43 which the alleles may have spread throughout dog breeds. Taken together, this work demonstrates that: 1) 44 the occurrence of low frequency alleles within breeds can reveal the influence of regional or functional 45 selection practices; 2) it is possible to trace the mode by which characteristics have spread across breeds 46 during historical breed formation; and 3) the necessity of addressing conflicting ideals in breed 47 descriptions relative to actual genetic potential is crucial. 48 49 Author Summary 50 From the sleek Doberman Pinscher to the coifed Poodle, the sunny Golden Retriever to the 51 aristocratic Pekingese, the world of purebred dogs offers options that appeal to nearly all aesthetics. Pure 52 dog breeds, of which there are over 400 worldwide, are created through selective breeding over multiple bioRxiv preprint doi: https://doi.org/10.1101/654343; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2 53 generations, toward an ideal goal of temperament, behavior, and physical appearance. Written 54 descriptions of these breed-specific ideals are produced and maintained by dedicated breed enthusiasts, 55 and provide guidelines that direct breeders in their selection choices. However, the genetic mechanisms 56 that produce the spectrum of canine colors and patterns, sculpt the small triangular ears of a Siberian 57 Husky or the long soft ears of a Basset Hound, are complicated and intertwined. This means that some 58 breeds can carry rare, hidden traits for many generations, hampering selection efforts toward uniformity. 59 We have determined the genotypes of >11,000 dogs, representing >200 breeds, at 12 genes that impact 60 coat color and physical characteristics. In doing so, we can now present realistic trait frequencies within 61 each breed, report occurrences of gene variants that can produce undesirable traits, and draw conclusions 62 about the historic spread of characteristics across modern related breeds and those with distant shared 63 ancestry. 64 65 Introduction 66 Guided by human selection, the domestic dog (Canis lupus familiaris) has become one of the 67 most physically diverse species, with hundreds of recognized breeds, differentiated from each other by 68 specific morphological and behavioral characteristics (1). One of the most readily accessible and easily 69 visualized defining characteristics of dog breeds is in their presentation of pigmentation and color 70 patterns. The nomenclature used currently to refer to dog pigmentation genetics was outlined by Little in 71 1957 (2). Scientific advancement since that time has allowed for the validation of many of his postulated 72 loci and the identification of causal genetic variants for many of the common coat colors in the species. 73 These coat color and trait variants are increasingly included in commercially-available genetic test panels, 74 the emergence of which has contributed greatly to the acceptance and implementation of genetic 75 screening of purebred dogs by owners, breeders, and veterinarians. In addition to providing valuable 76 information regarding the genetic status of potential breeding dogs in terms of disease state and 77 morphological traits, the combined genotype databases collected by these commercial entities provide a bioRxiv preprint doi: https://doi.org/10.1101/654343; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3 78 valuable resource for monitoring the diversity of selected alleles, such as those driving coat color, within 79 breeds. 80 The entirety of coat color patterns in mammals consist of spatial and temporal production of 81 phaeomelanin, yellow- to red-based pigment, and eumelanin, black- to brown-based pigment, controlled 82 by the interaction of multiple genes. In dogs, variation at the Agouti Signaling Protein (ASIP) gene 83 determines the distribution of eumelanin and phaeomelanin across the body of the dog and along the 84 individual hair shafts, as dictated by a four-allele dominance hierarchy: ay (fawn) > aw (wolf sable) > at 85 (tan points) > a (recessive black) (3–5). However, regardless of the accompanying ASIP genotype, the 86 Melanocortin 1 Receptor (MC1R) gene determines the ability of a melanocyte to produce eumelanin at 87 all, presenting its own dominance hierarchy of four alleles: EM (melanistic mask) > EG (grizzle/domino) > 88 E (wild type) > e (recessive red) (6–8). While the EM, EG, and E alleles all allow for the production of 89 both eumelanin and phaeomelanin, dependent on the pattern dictated by ASIP, homozygous inheritance of 90 e prevents eumelanin production, resulting in a completely phaeomelanin color (6–8). Further, the 91 dominant derived KB allele of Canine Beta-Defensin 103 (CBD103) prevents the production of patterning 92 by ASIP, resulting in a solid eumelanin phenotype when combined with a dominant MC1R genotype, or a 93 solid phaeomelanin phenotype when combined with a MC1R genotype of e/e (9). In this way, inheritance 94 of homozygous e at MC1R or dominant KB at CBD103 are epistatic to any of the ASIP phenotypes (8,9). 95 In addition to the dominant KB and recessive ky alleles of CBD103, an intermediate allele, kbr, produces 96 the brindle coat color pattern with alternating stripes of eumelanin and phaeomelanin (10). However, due 97 to the complex structural nature of the kbr allele, which has never been reliably defined and remains 98 unpublished, it was not possible to distinguish between it and KB for the purposes of this paper. 99 The base phaeomelanin and eumelanin pigments can be altered by a number of modifier genes, of 100 which Tyrosinase Related Protein 1 (TYRP1), Proteosome Subunit Beta 7 (PSMB7), Microophthalmia- 101 associated Transcription Factor (MITF), and RALY heterogeneous nuclear ribonucleoprotein (RALY) are 102 explored further in the scope of this paper. TYRP1 alters all eumelanin in hair and skin evenly from black 103 to brown, and presents as a compound heterozygote with at least four alternate recessive alleles: bs, bc, bd, bioRxiv preprint doi: https://doi.org/10.1101/654343; this version posted May 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4 104 and a variant that has thus far only been described in the Australian Shepherd (11–13). PSMB7 expression 105 produces the harlequin pattern of white, dilute, and deeply pigmented patches only when inherited along 106 with the merle phenotype of Premelanosome 17 (PMEL17, not tested in the present study) (14–16).