Chromatin accessibility and DNA methylation impact asymmetric subgenome dominance in allotriploid <i>Brassica</i> species

Polyploidy considerably influences eukaryotic evolution, often leading to structural and functional imbalances during the merging of subgenomes. Studies have demonstrated asymmetric subgenome dominance in polyploids, but the effects of accessible chromatin regions (ACRs) and DNA methylation-especially in resynthesized allotriploids-remain underexplored. In this study, we generated 2 Brassica allotriploid hybrids by crossing Brassica napus (AnAnCnCn) with Brassica rapa (ArAr). Among the 3 subgenomes (Ar, An, and Cn) in F1 hybrids, gene expression was highest in the An subgenome; however, the Cn subgenome exhibited greater dominance among homoeologous triplet genes (hGenes), which correlated with ACRs in proximal and genic regions of these dominant triplets. Variations in DNA methylation alone did not fully explain the subgenomic expression biases; however, RNA-directed DNA methylation pathway genes contributed to the differences in methylation levels. Mutants of BnaDCL3 and BnaRDR2 revealed the roles of their encoded proteins in regulating non-CG methylation through 24-nt siRNA interactions in the An and Cn subgenomes. We detected higher methylation levels in the gene bodies of Cn homologs in B. napus, regardless of their expression levels. Overall, this work reveals complex interactions among ACRs, DNA methylation, and subgenome dominance, advancing our understanding of polyploid genome regulation in resynthesized allotriploids.