Chicken shank color determined by Inhibition of dermal melanin (ID) is mediated by a structural variation regulating CDKN2A expression

It is well established that the color of a chickens shank is primarily determined by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses black pigmentation in the dermal layer of the shank, is the sole sex-linked mutation and its molecular mechanisms remained elusive. In this study, a resource family with segregating shank colors was constructed. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP located on the Z chromosome. Subsequent linkage mapping further refined the candidate region, enabling the screening of the candidate structural variation. The candidate structural variation is associated with the yellow shank and characterized by a 143 bp deletion accompanied by a 2 bp insertion. Within the same Topologically Associating Domain, only the CDKN2A gene showed differential expression. Functional studies, including CRISPR/Cas9-edited cells, provided evidence that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. The absence of melanocytes is likely due to their apoptosis. This study completes the puzzle of chicken shank color genetics and paves the way for the application of the ID mutation in the auto-sexing of chicks which is intensively needed in layer and broiler industries.