Two distinct structural variants involving EDN3 cause hyperpigmentation in chicken

Phenotypic diversity and its genetic basis are central questions in biology, with domesticated animals offering valuable insights due to their rapid evolution the last 10,000 years. In chickens, fibromelanosis (FM) is a striking pigmentation phenotype characterized by hyperpigmentation. A previous study identified a complex structural variant causing upregulated expression of the Endothelin 3 (EDN3) gene. However, the detailed structural arrangement and functional consequences of the variant remained unclear. In this study, we conducted a comprehensive genomic survey of 692 FM chickens representing 55 breeds and uncovered two distinct structural variants causing the FM phenotype: FM*A, the previously reported complex rearrangement involving the EDN3 locus1, and FM*B, a tandem duplication of a 16 kb region upstream of EDN3. We demonstrate that both structural variants (SVs) significantly upregulate EDN3 expression, with FM*B associated with even higher expression than FM*A. A luciferase reporter assays showed that the 16 kb region in FM*B contains powerful enhancers and the copy number expansion of this element is a likely explanation for EDN3 upregulation and hyperpigmentation. Furthermore, our analysis of linkage disequilibrium patterns allowed us to resolve the complex arrangement of duplications and inversion on the FM*A haplotype.