Understanding the molecular basis of phenotypic diversity is a critical challenge in biology yet we know little about the mechanistic effects of different mutations and epistatic relationships among loci that contribute to complex traits. interactions remain unknown. Here we identify protein-coding and cis-regulatory mutations in that underlie classical color phenotypes of pigeons and present a mechanistic explanation of their dominance and epistatic relationships. We also find unanticipated allelic heterogeneity at and allele is dominant to and is recessive to the others. Figure 1 Common color phenotypes of domestic rock pigeons Blue/black and brown phenotypes result from high amounts of eumelanin and low amounts of pheomelanin while melanin ratios are reversed in ash-red birds . In addition the autosomal recessive mutation (locus to elevate pheomelanin production generating red plumage color irrespective of locus P005091 genotype [2 8 (Fig. 1D). Mutant alleles of a third locus the sex-linked recessive (and to lighten plumage color and further enrich pigmentation diversity [1 2 8 (Fig. 1E-H). This detailed Mendelian understanding of key phenotypes provides a robust foundation to investigate how genes and alleles interact to generate color variation. However the molecular basis of this diversity – including the identities of genes underlying major pigmentation variants and a mechanistic explanation for their intra- and inter-locus interactions – remains unknown [9 10 Multiple mutations in underlie base color variation in pigeons Previously we reported whole-genome sequences for 41 rock pigeons  with diverse color phenotypes. To investigate the molecular identity of the color locus we compared the genomes of 6 ash-red to 26 blue/black pigeons for coding changes associated with pigmentation phenotypes using the Variant Annotation Analysis and Search Tool (VAAST) . A single gene achieved genome-wide significance: (= 1.3×10?6; Fig. S1A) which encodes a key enzyme in the melanin synthesis pathway. All blue/black pigeons were homozygous G on the sense strand at scaffold6:214991 (allele) while ash-red pigeons were hetero- or homozygous for C (allele) consistent with the dominant mode of inheritance of ash-red. The mutation causes an alanine-to-proline substitution at codon 23 (A23P) corresponding to the cleavage site of the signal peptide (Fig. 2A). In addition to finding a single haplotype containing the allele in our whole-genome panel (Fig. S1B) we found a perfect association between the dominant mutation and the ash-red P005091 phenotype in Mouse Monoclonal to Rabbit IgG. an additional 49 ash-red birds from 20 P005091 breeds and 105 blue/black or brown birds from 36 breeds (Fig. 2B). These results suggest that the ash-red mutation occurred only once and spread species-wide through selective breeding similar to our previous finding that the same mutation in underlies the head crest phenotype in multiple pigeon breeds . Figure 2 is the major color locus in domestic pigeons Quantitative RT-PCR analysis revealed that mRNA levels from developing feathers of and pigeons were indistinguishable (Fig. S1C); however the location of the mutation at the highly conserved cleavage site of the signal peptide (Fig. S1E) suggested that cleavage efficiency might be affected. We therefore expressed N- and C-terminal tagged and TYRP1 proteins in cell culture and found that cleavage efficiency was dramatically reduced by the mutation (relative efficiency: B+ = 1 ± 0.18 BA = 0.14 ± 0.04 n = 4 independent transfections each; < 0.002) (Fig. 2C). Furthermore spatial organization of pigment synthesis differed between genotypes: premelanosomes in regenerating feathers had a well-organized lamellar matrix and melanosomes were darkly pigmented while ash-red feathers (and feathers showed strongest staining localized to the limiting membrane of the melanosome (Fig. 2D) while staining was diffuse in melanosomes from and feathers. Thus the striking reduction in TYRP1 cleavage efficiency may disrupt the spatial organization of pigment synthesis activity providing insight into the molecular basis of dominance of the allele. The dominant (allele of mice a missense mutation near the same cleavage site causes melanocyte death probably through the accumulation P005091 of cytotoxic pigment intermediates . Whether a similar accumulation of cytotoxins contributes to the pheomelanic phenotype of ash-red pigeons is unknown. However unlike the.