Chicken Shank Color Determined by Inhibition of Dermal Melanin ( ID ) is Mediated by a Structural Variation Regulating CDKN2A Expression

Shank color in chickens is a classic quantitative trait governed by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses dermal melanogenesis in the shank, is the sole sex-linked mutation and its molecular mechanisms remain elusive. To identify the causal mutation, we established a resource population segregating for shank colors. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP on the Z chromosome. Linkage mapping subsequently narrowed the candidate region, within which we discovered a candidate structural variant associated with the yellow shank phenotype. This variant is characterized by a 143 bp deletion coupled with a 2 bp insertion. CDKN2A was the only gene within the same topologically associating domain to exhibit differential expression. Functional validation via CRISPR/Cas9-edited cells demonstrated that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. We propose that the resulting absence of melanocytes is likely due to apoptosis. This work resolves the molecular basis of the ID locus, thereby completing the genetic puzzle of chicken shank color. This discovery enables the development of molecular markers for auto-sexing of day-old chicks, a tool with significant potential for the poultry industry.