Abstract: Seed weight is a pivotal yield-determining trait in crops, and yet, the genetic and molecular mechanisms underlying its regulation in polyploid species remain underexplored. In a previous study, we identified cqSW.A03-2, a QTL that regulates thousand seed weight (TSW) in rapeseed (Brassica napus). Here, we identify BnaA3.AHK2, encoding a histidine kinase, as the causal gene of cqSW.A03-2. BnaA3.AHK2 enhances TSW through maternal control of seed coat cell expansion without significantly compromising other yield-related traits. Protein sequence divergence between parental haplotypes caused functional differentiation, with only the ZY50 allele showing functional kinase activity and rescuing developmental defects in Arabidopsis cytokinin receptor mutants. Strikingly, BnaA3.AHK2 seems to be a cytokinin-independent operator, contrasting with the canonical cytokinin signaling pathway. Transcriptome and protein interaction analyses reveal a signaling module where BnaA3.AHK2 engages BnaAHP–BnaARR phosphorelay components to regulate downstream targets. Notably, the favorable cqSW.A03-2 haplotype has been historically selected in modern breeding, and its introgression into elite hybrids boosted TSW by 3.6%–9.1%, demonstrating its breeding value. Our findings unveil a non-canonical signaling pathway for seed size regulation, providing a strategic genetic target to break yield trade-offs in polyploid crops.

Key words: BnaA3.AHK2, Brassica napus, cell size, seed size, thousand seed weight