Sequential Oligo‐ <scp>FISH</scp> reveals conserved synteny and rapid cytological diploidization in <i>Chrysanthemum</i> (Asteraceae) autopolyploids

The Chrysanthemum genus (Asteraceae) is a key polyploidy model, but its complex genomes obscure its origin and evolution. To address this, we developed chromosome-set-specific painting probes from the Chrysanthemum morifolium 'Zhongshanzigui' haploid genome, enabling precise identification of all nine chromosome sets. Combined with existing oligonucleotide probes (Oligo-Mix: CmOP-1 and CmOP-2), we established a novel sequential fluorescence in situ hybridization (FISH) procedure for comparative genomic analysis. Applying this across six Chrysanthemum species revealed extraordinarily conserved chromosomal synteny. Analysis of diploids (e.g. C. nankingense, C. lavandulifolium, and C. indicum) and their derived autotetraploids showed autopolyploidization involved amplification of large-scale repetitive sequences and loss of partial repeats. Crucially, rapid cytological diploidization (diploid-like bivalent pairing) occurred, associated with significant enrichment of repetitive sequences at meiotic crossover (CO) loci on homologous chromosomes. This leads us to hypothesize that repetitive DNA variation may facilitate precise chromosome segregation and diploid-like meiosis, thereby potentially ensuring polyploid stability. These findings provide essential tools for distinguishing homologous chromosomes and significant potential for elucidating homologous interactions to advance polyploid Chrysanthemum breeding.