Integrative Genomic and Transcriptomic Analysis to Identify Potential Candidate Genes Influencing Plumage Color Variation in Mule Ducks

Plumage color is a distinctive and economically important trait in poultry; however, its genetic architecture in hybrid species, such as the mule duck, remains poorly characterized. Traditional mapping approaches often fail to accurately establish causal genes, particularly for traits likely governed by major-effect loci. In this study, we used an integrated multi-omics approach to obtain a deeper understanding of the genetic basis of plumage color in mule ducks. Following whole-genome resequencing of 114 mule duck individuals, a combined analysis involving genome-wide association studies and genome scans for genetic differentiation ( F S T / π ) was conducted, which collectively highlighted the EDNRB2 genomic region. Comparative transcriptomics of black versus white feather follicles confirmed that EDNRB2 and key melanogenesis genes were significantly upregulated. This convergence of genomic and transcriptomic evidence was further confirmed by experimental validations. Quantitative polymerase chain reaction confirmed differences in EDNRB2 expression levels, while competitive allele-specific PCR (KASP) genotyping in an independent population revealed a significant association between specific upstream SNPs of EDNRB2 and plumage color scores. Our study revealed EDNRB2 as a pivotal candidate gene regulating plumage color variation in mule ducks. The multi-tiered approach, from population-level genomics to molecular validation, identified a key genetic mechanism, and also provided functionally validated KASP markers, establishing a direct foundation for further developing mule duck breeding in the poultry industry.