Two near-complete assemblies reveal R-subgenome structural and centromeric divergence associated with reproductive isolation in hexaploid triticale.

We present two near-complete triticale genome assemblies and perform pan-centromere analyses across >200 haplotypes from parental species, synthetic allopolyploids, cultivars, and 337 resequenced accessions, revealing a shared chromatin framework underlying functional centromeres in all three subgenomes, where wheat- and rye-derived CENH3 co-occupy divergent centromeric repeats. Postpolyploid evolution drives extensive centromere remodeling, including size convergence, repeat turnover, and locus repositioning, with the R subgenome emerging as the primary source of structural and centromeric divergence. In F1 hybrids, R-subgenome structural and centromeric divergence is associated with meiotic abnormalities, including chromosome misalignment, lagging chromosomes, and micronuclei formation, suggesting a link between centromere variation and reduced meiotic stability.