Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

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We thank the numerous horse owners who provided samples, T. Raudsepp and I. Randlaht for samples from Estonian native horses, C. Asa and M. Fischer (St. Louis Zoo) and F. Marshall for providing hair samples from the Somali wild ass, W. Zimmerman for photographs of Przewalski's horse, S. Fard, H. Ring and F. Hallböök for advice on histological characterization, O. Ryder, L. Chemnick and C. Steiner for delivering DNA extracts from Przewalski's horses, C. Der Sarkissian and L. Ermini for assistance in whole-genome resequencing at the Centre for GeoGenetics, Denmark, and the HudsonAlpha Genomic Services Laboratory for RNA-seq. This work was supported by grants from the Knut and Alice Wallenberg foundation (to L.A.) and the US National Institutes of Health (to G.S.B.), as well as by an Erasmus Mundus fellowship within the framework of the European Graduate School of Animal Breeding and Genetics (to D.S.). Sequencing was performed by the SNP&SEQ Technology Platform, supported by Uppsala University and Hospital, the Science for Life Laboratory and the Swedish Research Council (80576801 and 70374401).

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Author notes

    • Freyja Imsland
    •  & Kelly McGowan

    These authors contributed equally to this work.


  1. Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.

    • Freyja Imsland
    • , Carl-Johan Rubin
    • , Elisabeth Sundström
    • , Jonas Berglund
    • , Kerstin Lindblad-Toh
    •  & Leif Andersson
  2. HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.

    • Kelly McGowan
    •  & Gregory S Barsh
  3. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.

    • Kelly McGowan
    • , Corneliu Henegar
    •  & Gregory S Barsh
  4. Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.

    • Doreen Schwochow
    • , Ulla Gustafson
    • , Gabriella Lindgren
    • , Sofia Mikko
    •  & Leif Andersson
  5. Institut National de la Recherche Agronomique (INRA), AgroParisTech, Génetique Animale et Biologie Intégrative, Jouy-en-Josas, France.

    • Doreen Schwochow
  6. Menntaskólinn við Hamrahlíð, Reykjavík, Iceland.

    • Páll Imsland
  7. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

    • Kerstin Lindblad-Toh
    •  & Claire Wade
  8. Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, Davis, California, USA.

    • Lee Millon
    •  & Maria Cecilia T Penedo
  9. Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

    • Mikkel Schubert
    •  & Ludovic Orlando
  10. Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, USA.

    • Leif Andersson


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L.A. led the genetic characterization and G.S.B. led the RNA-seq and immunohistochemistry studies. F.I., P.I., K.M. and M.C.T.P. did the sampling. F.I. was responsible for phenotyping and carried out genotyping together with U.G., L.M. and M.C.T.P. K.M. performed immunohistochemistry analysis. C.H. performed RNA-seq analysis. C.-J.R., F.I., J.B., M.S. and L.O. were responsible for genome sequence analysis. E.S. analyzed transcription factor binding sites. D.S. contributed to TBX3 expression analysis. F.I., L.A., M.C.T.P., K.L.-T., G.L., S.M. and C.W. took part in the initial mapping of Dun. L.A., F.I., K.M., C.H. and G.S.B. wrote the manuscript with input from other authors. All authors approved the manuscript before submission.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gregory S Barsh or Leif Andersson.

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    Supplementary Figures 1–6 and Supplementary Tables 1, 2, 4 and 7.

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    Supplementary Table 3: Summary of results of SNP screen using 384 SNPs from the TBX3 region.

    Genotypes of the SNPs most strongly associated with the dun and non-dun phenotypes, marked by red dots in Figure 2e. The results of three different association tests for dominant genotypes are reported: (i) Dun versus non-dun1 including both domestic and Przewalski's horses, (ii) Dun versus non-dun1 including only domestic horses and (iii) Dun versus non-dun (non-dun1 and non-dun2 combined) including both domestic and Przewalski's horses. All but four SNPs could be excluded as being causative on the basis of the genotypes of Przewalski's horses. Two of those four could also be excluded on the basis of the presence of a recombinant Dun haplotype in domestic horse 7. The genotype was confirmed via Sanger sequencing in this individual and in two related dun horses that also proved recombinant. "nc" indicates no call.

  2. 2.

    Supplementary Table 5: Haplotype analysis of the 40-kb region centered at the non-dun2 deletion on the basis of the SNP screen.

    Individuals shown are representative for the data set. "nc" indicates no call.

  3. 3.

    Supplementary Table 6: Detailed results of the RNA-seq differential gene expression study in skin from dun and non-dun horses.

    (a) Samples obtained from the croup. (b) Samples obtained from the dorsal midline. Four columns are included for each detected gene, displaying the average overall expression level (normalized gene counts) and a log2-transformed fold change between dun and non-dun samples, as well as the degree of statistical significance of the observed differences expressed as P values and q values (adjusted for multiple testing using the Benjamini-Hochberg correction approach). Genes are sorted according to the observed P values in the analysis of croup samples.

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