Association of a synonymous PMEL variant with localized melanin deposition in chicken tail feathers

Plumage coloration is an economically important trait in poultry. Although PMEL mutations cause systemic pigmentation defects in vertebrates, their role in localized melanin deposition remains unclear. Wuhua yellow chickens, exhibiting black-spotted (BS) tails on a white background, provide a model system to address this knowledge gap. We integrated whole-genome sequencing of 47 roosters (24 solid white [SW] and 23 BS) with data from 100 black-tailed samples and transcriptomic profiling of follicles. Genome-wide association analysis identified 38 significant SNPs on chromosome 33 within PMEL and related genes, overlapping with selective sweep regions. Transcriptomics revealed 2702 differentially expressed genes, with PMEL significantly upregulated in BS follicles, which was confirmed by quantitative real-time PCR. Gene set enrichment analysis highlighted melanogenesis pathways (GO:0043473; normalized enrichment score = 1.81, false discovery rate = 0.037), with PMEL as a core gene. Kompetitive allele-specific PCR genotyping of the synonymous PMEL variant (chr33:7,093,765) showed a near-perfect genotype-phenotype concordance in males: SW were AA (476/476), BS were predominantly GA (98/115), and black-tailed breeds were GG (107/107). Critically, genotyping of 315 SW hens identified 14 G-allele carriers, confirming cryptic carriers and sex-dimorphic expression. The A allele was absent in diverse populations but present in commercial broilers (A = 0.105) according to the Global Chicken Reference Panel database. These results indicate that the G allele is strongly associated with melanin deposition, with the GA genotype linked to localized spotting and GG to systemic pigmentation. This study provides multi-omics insights into avian pigmentation and offers a kompetitive allele-specific PCR marker for precision breeding.