Dog colour patterns explained by modular promoters of ancient canid origin

Abstract

Distinctive colour patterns in dogs are an integral component of canine diversity. Colour pattern differences are thought to have arisen from mutation and artificial selection during and after domestication from wolves but important gaps remain in understanding how these patterns evolved and are genetically controlled. In other mammals, variation at the ASIP gene controls both the temporal and spatial distribution of yellow and black pigments. Here, we identify independent regulatory modules for ventral and hair cycle ASIP expression, and we characterize their action and evolutionary origin. Structural variants define multiple alleles for each regulatory module and are combined in different ways to explain five distinctive dog colour patterns. Phylogenetic analysis reveals that the haplotype combination for one of these patterns is shared with Arctic white wolves and that its hair cycle-specific module probably originated from an extinct canid that diverged from grey wolves more than 2 million years ago. Natural selection for a lighter coat during the Pleistocene provided the genetic framework for widespread colour variation in dogs and wolves.

Fig. 1. Coat patterns controlled by ASIP.

Drawings of the five pattern types caused by ASIP regulatory variation are shown on the left, with representative photographs shown on the right. A completely black coat caused by homozygosity for ASIP loss-of-function is not shown. Within each pattern type, there may be variation due to other factors including: (1) the position of the boundaries between pheomelanic and eumelanic areas, for example in black saddle or black back; (2) the shade of pheomelanin (red to nearly white); (3) presence of a black facial mask or white spotting caused by genes other than ASIP; and (4) length and/or curl of hair coat. Patterns are displayed in order of dominance.

Fig. 2. Structural variation at the ASIP locus in domestic dogs with different colour patterns.

a, Genomic context (NCBI annotation release 105, CanFam3.1 assembly). Numbers in blue indicate previously reported variant associations—i (ref. ), ii (ref. ) and iii (ref. )—referred to in the Discussion and in Extended Data Fig. 2. b, The canine ASIP gene has three alternative promoters and 5′-non-coding exons (nucleotide coordinates denote their 3′-ends. Structural variation within 1.5-kb sections of the ventral- and hair cycle-specific promoters explains five different colour patterns in dogs. Two different VP haplotypes and five different HCP haplotypes are schematically indicated. The asterisk represents the third promoter and non-coding exon that is not related to ASIP pattern variation as described in the text and Fig. 1. c, Summary of how extended haplotype combinations are related to colour pattern phenotypes. Semiquantitative ASIP expression levels are depicted with one or two arrows or an X for no expression (Extended Data Fig. 1).

Fig. 3. A family of dogs segregating dominant yellow and two black back haplotypes.

Extended haplotype combinations in a family of Chinook dogs illustrating that, in combination with VP2, HCP3 and HCP5, both confer a black back phenotype.

Fig. 4. Yellow dogs and white wolves share an ancient HCP haplotype.

a, Genotypes at 377 SNVs (columns) at the ASIP locus in grey wolves and dogs (rows), coded for heterozygosity (light blue), homozygosity for the reference (yellow) or the alternate (dark blue) allele or as missing genotypes (white). Alternate first exons (arrows) and nearby DY-associated structural variants (SINE insertions, green; polynucleotide expansions, orange) are included for reference. b, Maximum likelihood phylogenies, including seven extant canid species and the dog, from 48- and 16-kb intervals upstream or downstream of the HCP, respectively. Grey wolf/dog phyletic clades are highlighted with boxes to indicate relationships that are consistent (blue) or inconsistent (red) with genome-wide phylogenies. c, Images of a grey wolf, Arctic grey wolf and Tibetan wolf. d, A phylogeny representing distinct HCP evolutionary histories inferred from genetic variation in extant canids. Structural variants (as represented in Fig. 2) and derived SNVs (cyan) distinguish wolf-like canid (blue), ghost lineage (red) and basal canid (black) haplotypes.

Fig. 5. Distribution of ASIP alleles in ancient dogs and wolves, and an evolutionary model for dominant yellow acquisition.

a, ASIP haplotypes were inferred from WGS of five ancient dogs (circles), two ancient wolves (squares) and 68 modern wolves (pie charts) distributed across the Holarctic (Extended Data Fig. 7 and Supplementary Table 12 show detailed haplotype representations). Asterisks indicate SY/DY haplotypes for which the HCP1 insertion is either absent (SY*) or not ascertainable (DY*). b, A model for the origin of the dominant yellow haplotype and its transmission into dogs and Arctic wolves, in which molecular alterations at modular promoters were acquired by introgression (red, HCP1) or by mutation in the grey wolf (blue, VP1). The timelines for speciation events, dog domestication and geological events affecting grey wolf dispersal are based on prior studies^,.

Extended Data Fig. 1. Relative transcription of promoter variants.

RNA-seq counts (transcripts per million reads, TPM) from dorsal (D) and ventral (V) regions of two dominant yellow, three black back and four agouti dogs obtained from skin biopsies as described in Methods and in Supplementary Table 8. HCP samples also included a black saddle dog. Black dots are from RNA-seq data and grey dots are from STRT RNA-seq data.

Extended Data Fig. 2. Dog haplotypes across the ASIP locus with comparison to previously associated variants for colour patterns.

Dog coat pattern phenotypes are listed on the left. The genomic organization of the ASIP gene with its alternative promoters is illustrated at the top. Yellow indicates a homozygous match to the reference genome, grey deleted, white heterozygous and blue homozygous alternate allele. The black rectangles highlight the promoter regions. Green triangles represent the location of variants that were previously identified to distinguish different alleles for coat colour patterns: (i) The previously identified intronic duplication, “RALY dup”, associated with BS vs. BB haplotypes in some breeds, lies 86 kb to the left of the VP but recombinants (Supplementary Table 7) exclude a causal role for ASIP pattern variation. Similarly, (ii), a SINE insertion associated with BB and BS haplotypes in some breeds and, (iii), missense variants in exon 4 associated with DY haplotypes, are also excluded from a causal role in ASIP pattern variation by rare recombinants (Supplementary Table 7). In the samples presented here, the dominant yellow haplotype extends through the coding sequence where the missense variants associated with this haplotype were previously identified10. The results shown here will allow more accurate genetic testing in the future. Samples used are listed in Supplementary Table 3. Raw genotyping results are in Supplementary Table 4 and summary results comparing previously identified variants are in Table 1 and Supplementary Table 7.

Extended Data Fig. 3. Expanded canid phylogenetic tree inferred from 48-kb region including the ventral promoter.

An expanded version of the maximum likelihood tree shown in Fig. 4b, with 34 canids, representing 7 of 9 extant species.

Extended Data Fig. 4. Expanded canid phylogenetic tree inferred from 16-kb region within and downstream of the hair cycle promoter.

An expanded version of the maximum likelihood tree shown in Fig. 4b, with 34 canids, representing 7 of 9 extant species.

Extended Data Fig. 5. Genomic distribution of derived substitutions across the ASIP locus.

a, Canid phylogenies for the ventral (48 kb) and hair cycle (16 kb) promoter regions, with relevant internal branches marked by the occurrence of derived variants plotted in (b). b, Derived substitutions shared by grey wolf and dogs (cyan). Ancestral alleles on DY/Arctic wolf haplotypes (red) or BB and DY/Arctic wolf haplotypes (orange) that correspond to derived substitutions among the core group of wolf-like canids (Supplementary Table 12). The broken lines demarcate the HCP region (chr24:23,375,800–23,380,000). The solid line signifies the downstream boundary for phylogenetic analysis. The solid green and orange lines indicate the positions of the SINE and 24 bp insertion, respectively, associated with the DY/Arctic wolf haplotype.

Extended Data Fig. 6. The distribution of ASIP haplotypes in modern grey wolves.

Modern grey wolves (squares) from (a) North America (n = 45) or (b) Eurasia (n = 23) were genotyped for 5 structural variants and 6 SNVs using whole genome sequencing data. Wolves are coloured by inferred VP and HCP haplotypes, as indicated in the figure legend and in Supplementary Table 11. The asterisk indicates an SY-like haplotype without the HCP1 insertion.

Extended Data Fig. 7. Evolutionary diversification of ASIP haplotypes observed in grey wolves and dogs.

The colour (red or blue) of ASIP haplotype segments indicates ancestral species of origin, inferred from phylogenetic analysis (Fig. 4b, Extended Data Figs. 3, 4). Relevant structural variants near the ventral (VP) and hair cycle (HCP) promoters are depicted as yellow triangles (polynucleotide expansions), green bars (SINE insertions) and white bars (deletions). Modified promoter activity is indicated by an X mark (no activity) or an additional arrow (elevated expression), based on RNA-seq (Extended Data Fig. 1) and/or inference from coat colour (Figs. 1, 2c, 4c).