A cis-regulatory mutation in the EDN3 promoter drives coordinated formation of comb and skin color in Anyi gray chickens.

Comb and skin pigmentation are key external traits in domestic chickens, closely correlated and serving as important visual cues for consumers. The Anyi Gray chicken, an indigenous breed from Jiangxi Province, China, exhibits two stable phenotypes: red comb with white skin (RAY), and purple-gray comb with purple-gray skin (PAY). Although skin color is completely linked to comb color, the genetic basis of this coordination remains unclear. We performed whole-genome resequencing (∼ 10 × depth) on RAY and PAY individuals, followed by a case-control genome-wide association study (GWAS). A highly significant variant, g.10818413 T > C, located in the promoter region of EDN3, was identified (P < 4.065343e-09). Transcription factor binding site prediction revealed that this mutation disrupts an NFAT5 binding site. Genotyping of 97 Anyi Gray chickens showed complete concordance between genotype and phenotype: all PAY individuals carried the TC genotype, while all RAY individuals had the TT genotype; no CC genotypes were detected. Validation in an F1 hybrid population (n = 61) and in 140 individuals from other Jiangxi native breeds confirmed the absence of the CC genotype. Functional assays demonstrated that NFAT5 negatively regulates EDN3 transcription. Dual-luciferase reporter assays confirmed NFAT5 binding to the EDN3 promoter and its suppressive effect. Tissue expression analysis showed significantly higher EDN3 expression in the comb, skin, and chest muscle of PAY individuals compared to RAY individuals, reinforcing the phenotypic linkage. These findings indicate that the EDN3 g.10818413 T > C mutation reduces NFAT5 binding affinity, cis-regulating EDN3 transcription and driving the coordinated pigmentation of the comb and skin. This study reveals a shared regulatory mechanism underlying the phenotypic correlation between comb and skin pigmentation in Anyi Gray chickens.