The Mottling Phenotype in Chickens Shows Genetic Heterogeneity and Is Caused by Mutations at the EDNRB2 Locus

Mottling is a widespread intra-feather pattern present in many breeds of domestic chickens worldwide and inherited as an autosomal recessive trait. An amino acid substitution Arg332His in the EDNRB2 gene was previously reported as responsible for the mottling phenotype in several Japanese breeds of chickens. Here, we show that this substitution is not causal for mottling in Mottled Houdan chicken, a breed that originated in France. A strong association between a 230 kb region harboring the EDNRB2 gene and the mottling phenotype was documented by linkage mapping. After the screening of SNPs, indels, and structural variants in this region via whole genome sequencing, a missense mutation Ala228Thr of EDNRB2 showed complete association in Mottled Houdan, and three other mottling breeds. It was also associated with the mottled phenotype in an experimental chicken line segregating for the phenotype. Furthermore, we documented significant allelic imbalance of EDNRB2 in heterozygous birds, implying that regulatory mutation(s) may also contribute to the mottling phenotype. However, no single mutation showed perfect concordance with the mottling phenotype in all mottled breeds, providing evidence for genetic heterogeneity at this locus. We propose that there are at least three different alleles responsible for mottling in chickens. Our results provide an example of parallel evolution of mottling alleles in different breeds of domestic chicken.