Abstract

While CYCLOIDEA2 (CYC2) genes regulate floret identity and bilateral symmetry in Asteraceae capitula, the molecular mechanisms underlying coordinated regulation among clustered CYC2 genes remain unclear. We investigated how tandemly arrayed CYC2 genes function cooperatively to control ray floret development in marigold (Tagetes erecta). Using a natural tubularized ray floret mutant, we integrated bulked segregant RNA-seq (BSR-seq) analysis, transcriptome profiling, transgenic validation, and molecular interaction assays to dissect the regulatory mechanisms among clustered CYC2 genes. Five CYC2 genes (TeCYC2b, TeCYC2c, TeCYC2e1, TeCYC2e2, and TeCYC2g) are tandemly arrayed on chromosome 2, forming an integrated transcriptional module. A MYB recognition element insertion in the TeCYC2c promoter suppresses its expression, disrupting ray floret identity. TeCYC2c functions as a hub protein that recruits TeCYC2b, TeCYC2e1, and TeCYC2g through heterodimeric protein-protein interactions, facilitating TeCYC2e1-mediated transcriptional activation of TeCYC2e2. Comparative analysis across 18 Asteraceae species reveals extensive CYC2 copy number variation (1-28 genes) with prevalent gene clusters (14/18 species). CYC2 gene clusters operate as integrated regulatory networks rather than independent units, with cis-regulatory evolution driving functional specialization in ray floret development. These findings provide a molecular framework for understanding inflorescence diversification in Asteraceae.