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Modern sugarcane cultivars are highly polyploid and derived from the hybridization of Saccharum officinarum and S. spontaneum, thus leading to singularly complex genomes. The complex genome has hindered the study of genomic structures. Here, we adopted a computational strategy to isolate highly repetitive and abundant sequences in either S. officinarum or S. spontaneum and isolated four S. spontaneum-enriched retrotransposons. Fluorescence in situ hybridization (FISH) assays with these repetitive DNA sequences generated whole-genome painting signals for S. spontaneum but not for S. officinarum. We demonstrated that these repetitive sequence-based probes distinguish the parental S. spontaneum genome in hybrids derived from crosses between it and S. officinarum. A cytological analysis of 14 modern sugarcane cultivars revealed that the percentages of chromosomes with introgressive S …