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Chromosome-specific identification is a powerful technique in the study of genome structure and evolution. However, there is no reliable cytogenetic marker to unambiguously identify each of the chromosomes in sugarcane ( Saccharum  spp., Poaceae), which has a complex genome with a high level of ploidy and heterozygosity. In this study, we developed a set of oligonucleotide (oligo)-based probes through bioinformatic design and massive synthetization. These probes produced a clear and bright single signal in each of the chromosomes and their eight homologous chromosomes in the ancient species  Saccharum spontaneum  (2 n  = 8 x  = 64). Thus, they can be used as reliable markers to robustly label each of the chromosomes in  S. spontaneum . We then obtained the karyotype data and established a nomenclature based on chromosomal sizes for the eight chromosomes of the octoploid  S. spontaneum . In addition, we also found that the 45S and 5S rDNAs demonstrated high copy number variations among different homologous chromosomes, indicating a rapid evolution of the highly repeated sequence after polyploidization. Our fluorescence  in situ  hybridization (FISH) assay also demonstrated that these probes could be used as cross-species markers between or within the genera of  Sorghum  and  Saccharum . By comparing FISH analyses, we discovered that several chromosome rearrangement events occurred in  S. spontaneum , which might have contributed to the basic chromosome number reduction from 10 in sorghum to 8 in sugarcane. Consistent identification of individual chromosomes makes molecular cytogenetic study possible in sugarcane and will facilitate fine chromosomal structure and karyotype evolution of the genus  Saccharum .

frontiers in plant science

Comprehensively Characterizing the Cytological Features of Saccharum spontaneum by the Development of a Complete Set of Chromosome-Specific Oligo Probes