Ardaman, A.; Forgiarini, C.; Arunkumar, R.

Intraspecific hybridization in allopolyploid plant genomes has the potential to induce non-additive changes in gene expression and DNA cytosine methylation, partly through interactions among divergent parental subgenomes. However, the extent to which intraspecific hybridization reshapes gene expression, coordinates homoeolog regulation, and remodels methylation in higher-order polyploids remains poorly quantified. To address this, we sequenced seedling leaf transcriptomes and methylomes from two parental cultivars of hexaploid bread wheat (Triticum aestivum L.) and their hybrids. More than 40% of genes were differentially expressed between hybrids and parents, although many were not differentially expressed between the parents themselves, consistent with complex trans-regulatory effects in the hybrid genome. This effect was more pronounced for homoeologs whose relative expression differed between the parents. These expression shifts often occurred simultaneously across all three homoeologs within triads, reducing homoeolog expression bias (HEB) in the hybrids. CG methylation levels were similar between the parents and hybrids in regions of low genetic divergence and in transposable element (TE)-rich regions, whereas CG sites in gene-rich regions showed more additive inheritance (hybrids intermediate between parents), particularly when parental haplotypes were themselves divergent. TE and gene body methylation (gbM) was strongly conserved in parents and hybrids. gbM was associated with more balanced homoeolog expression and fewer non-additive expression changes. CHH methylation showed overdominance, whereas non-conserved CHG methylation was enriched in TE-rich regions, suggesting that non-CG remodeling may reflect parental differences in TE and small-RNA content. Our results show that intraspecific hybridization within a hexaploid species can generate non-additive changes in gene expression and DNA methylation in seedling leaf tissue, while the presence of homoeologous genes, parental HEB, parental genetic and methylation divergence, and genomic location have varying levels of influence on expression or methylation remodeling.