Research note: Systematic evaluation reveals the genetic independence of atypical shank feathering from PITX1, TBX5, and CUBN variants in chickens.

Shank feathering (ptilopody) in chickens is primarily driven by cis-regulatory variants at the PITX1, TBX5, and CUBN loci. Recent refinement has pinpointed the major PITX1 determinant to an ∼18-kb deletion adjacent to H2AFY, which suppresses distal gene expression by disrupting a chromatin insulator. To evaluate these variants, we surveyed over 20,000 birds from diverse populations and identified an atypical, mild-to-variable shank-feathering phenotype in five Chinese indigenous breeds and one selected line. This phenotype is characterized by sparse, short feathers that do not produce the classic "booted" appearance and has at a low prevalence (0.94%-4.83%). The trait showed no significant association with sex (P > 0.05), ruling out a sex-linked inheritance pattern. Targeted genotyping of the known major-effect variants, including the refined H2AFY deletion, was performed in 153 atypical birds, 190 clean‑legged controls, and representative heavy/moderate-feathered individuals. Our results reveal a widespread genetic decoupling from established models in atypical cohorts. In Xiangdong chickens, 38.1% of feathered individuals lack any of the known mutations including PITX1/H2AFY, TBX5, and CUBN. The TBX5 variant exhibited significant incomplete penetrance (66.7%) and failed to co-segregation with the trait in Xiangdong chickens (P > 0.05). No additive phenotypic effects were observed in individuals harboring multiple variants (Cochran-Armitage trend test, P = 0.842), and the CUBN variant showed no association across the studied populations (P > 0.05). These findings provide quantitative evidence that atypical ptilopody operates under a genetic architecture independent of the established PITX1-TBX5 variants. Our results suggest greater genetic complexity than previously recognized and underscore the potential for discovering alternative regulatory mechanisms within these indigenous populations.