A regulatory 24-bp insertion at the BCO2 gene region is associated with yellow skin in chickens

Chicken skin colour is an economically important trait influenced by consumer preferences across different markets. Previous studies established that white skin is dominant over yellow skin, with the β, β-carotene-9',10'-dioxygenase (BCO2) identified as the candidate gene. However, the precise causal mutation within this gene region has remained unidentified for over a decade, limiting the development of reliable molecular markers for breeding programs.

Through genome-wide association analysis of 381 chickens, we confirmed that the BCO2 gene region on chromosome 24 was the major locus associated with skin colour. Transcriptome analysis revealed approximately 590-fold higher BCO2 expression in white-skinned than yellow-skinned chickens. By integrating whole-genome sequencing data from 63 individuals with high-quality genome assemblies, we identified a 24-bp insertion that showed near-complete co-segregation with the yellow skin phenotype in a backcross family of 180 individuals. Electrophoretic mobility shift assays revealed an insertion-dependent DNA-protein interaction, and DNA pull-down coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) prioritised lymphoid enhancer-binding factor 1 (LEF1) as a leading candidate interactor. Chromatin profiling revealed elevated trimethylation of histone H3 at lysine 27 (H3K27me3) and reduced chromatin accessibility at the gene locus in yellow-skinned chickens. Functional experiments revealed that knockdown of LEF1 or inhibition of enhancer of zeste homologue 2 (EZH2) significantly increased BCO2 expression, supporting a model in which the insertion is associated with LEF1/EZH2-sensitive, polycomb repressive complex 2 (PRC2)-linked repression and transcriptional silencing.

The 24-bp insertion in the BCO2 regulatory region is a candidate causal variant for yellow skin in chickens, refining the genetic model of chicken skin colour variation and supporting a LEF1-PRC2-associated regulatory mechanism potentially involved in avian carotenoid metabolism. This finding enables the development of a reliable PCR-based diagnostic marker for skin colour, facilitating marker-assisted selection in poultry breeding and providing new insights into the genetic regulation of avian pigmentation.