Abstract
Centromeres are essential for chromosome segregation, yet their repetitive nature makes them among the most difficult genomic regions to assemble and interpret. In polyploid plants, centromeres provide a unique record of chromosome evolution because centromeric repeats can be reshaped by polyploidization, subgenome differentiation and chromosomal rearrangement. Here, we combined low-coverage repeatome profiling, genome-wide mapping, fluorescence in situ hybridization (FISH), fiber-FISH and higher-order repeats (HORs) analysis to investigate centromere-associated repeats in Miscanthus sinensis (2n = 2x = 38). We identified MsSat1, a 137 bp satellite repeat representing approximately 2.1% of the genome, as a major centromere-associated satellite repeat. MsSat1 signals overlapped with the Saccharum officinarum centromeric retrotransposon probe and formed continuous tandem arrays on DNA fibers, supporting its association with centromeric domains. MsSat1-based clustering further recovered 13 related satellite variants, many of which showed strongly biased distribution between the A and B subgenomes. Among them, MsSat1-G displayed the most striking pattern, with strong B-subgenome enrichment and chromosome 7-specific localization. Detailed analysis revealed that MsSat1-G was embedded within an expanded MsSat1-rich domain on the fusion-derived chromosome 7, where it showed a more heterogeneous HORs architecture than the major MsSat1 array. These findings suggest that chromosome fusion was accompanied by local centromeric satellite amplification and repeat-architecture remodeling. Together, our study reveals that fusion-derived centromeres can preserve distinct satellite signatures and provides cytogenetic and genomic evidence that centromeric repeats record both subgenome differentiation and chromosome restructuring in M. sinensis.