Recurrent tandem duplication of a conserved enhancer underlies parallel genetic origins of rumplessness in chickens

Abstract

Congenital caudal body truncation in chickens—the rumpless phenotype—provides a powerful model for studying vertebrate axis elongation. This trait has been linked to misexpression of IRX1 and IRX2 in the embryonic tail bud, and breed-specific candidate mutations, including a deletion upstream of IRX1 proposed for the Piao breed, have been reported, yet whether the various breeds share a common causal basis has remained unresolved. By combining whole-genome resequencing, long-read sequencing, and population genomic analyses of four geographically isolated rumpless breeds (Chinese Piao, Japanese Uzurao, Belgian Barbu de Grubbe, and Chilean Araucana), we identify distinct tandem duplications of the IRX1/IRX2 intergenic region that converge on a single ∼700 bp conserved noncoding element, which we designate EnhRp. Haplotype and breakpoint analyses reveal that these duplications arose through at least two mutational events rather than a single shared origin. Functional assays in chicken embryos demonstrate that duplicating EnhRp acts as a hypermorphic gain-of-function mutation, elevating enhancer activity in the tail bud in a dose-dependent manner, consistent with the ectopic IRX1/IRX2 expression. Our findings support a model of parallel evolution in which distinct structural variants recurrently target a specific cis-regulatory element across at least two independent lineages, highlighting the critical role of noncoding variation in shaping morphological diversity.