High-Definition Cytogenetic Mapping of modern sugarcane cultivars by Oligo-FISH

Sugarcane ( Saccharum spp.), a dual-purpose crop valued for sugar and energy production, serves as an important industrial raw material and has substantial economic impact. However, its complex polyploid genome, characterized by highly variable chromosomal composition due to interspecific hybridization between S. officinarum and S. spontaneum , renders the detection of chromosomal rearrangements elusive. Here, we investigated the genome architecture of sugarcane cultivars using fluorescence in situ hybridization (FISH) with chromosome-specific painting probes ( S. officinarum ) and repetitive sequence probes ( S. spontaneum ). High-resolution karyotyping revealed extensive variation in chromosome numbers and structures of sugarcane. Analysis revealed lineage-specific chromosomal rearrangements, including high-frequency translocations exclusively associated with S. spontaneum genomic backgrounds. Molecular cytogenetic maps achieved single-chromosome resolution, enabling precise tracking of translocation fragments. Notably, chromosome-specific polymorphism patterns were uncovered, with chromosomes 4 and 10 exhibiting elevated susceptibility to rearrangements. These findings provide a well-characterized profile of the potential chromosomal rearrangements for each nonhomologous chromosome, and even homeologous chromosomes between S. officinarum and S. spontaneum . The established cytogenetic system will serve as a diagnostic toolkit for tracing chromosomal inheritance in sugarcane breeding programs, and lay a foundation for investigating the mechanistic basis of chromosome translocations. • Pioneered a precision chromosome identification system for modern sugarcane cultivars. • Decoded chromosomal variation events in modern sugarcane cultivars. • Quantified the proportion of chromosomal introgression in modern sugarcane cultivars. • Constructed high-resolution of modern sugarcane cultivars cytogenetic maps for reference.