Tandem duplication upstream of HSF5 associates with transcript remodeling and triple-comb morphology in chickens.

The genetic basis of the triple comb in chickens remains unknown. Here, we integrated population genomics, genome-wide association studies, structural variant analysis, long-read sequencing, and embryonic transcriptomics in Wuhua Yellow chickens. We identified a 15.7-kb tandem duplication upstream of HSF5 on chromosome 19 as the primary candidate. At embryonic day 8, overall HSF5 expression was unchanged, yet we observed localized exon upregulation within the duplicated region, a dosage-dependent expression gradient, and extensive non-canonical splicing. Public epigenomic data further indicate baseline enhancer activity in this interval. Copy number correlated positively with comb prong number (R 2 = 0.45, adjusted p = 9.26 × 10-7). Exploratory fertility assessments revealed a lower fertilization rate in triple-comb crosses, suggesting a potential pleiotropic cost. These findings strongly implicate HSF5 as a key candidate and demonstrate that a tandem duplication associates with phenotypic innovation through transcript remodeling rather than simple gene misexpression.