Background Repetitive sequences, including tandem repeats and transposable elements (TEs), are prominent components of eukaryotic genomes, each of them possesses distinct characteristics, evolutionary trajectories, and mechanisms of origin and amplification. Previous studies have suggested potential connections between these repetitive sequences. For instance, Cassandra elements, a member of the terminal repeat retrotransposons in miniature (TRIM) group within the TEs, may have originated from 5S rDNA. Nevertheless, a comprehensive understanding of their genomic landscapes and evolutionary trajectories within complex genomes of diverse plant species, characterized by varying ploidy levels, chromosome numbers, subgenomes, and structural variations remains limited. Results In this study, we conducted a comparative analysis of 5S rDNA and Cassandra elements in sugarcane and its close relatives to characterize their genomic landscapes and evolutionary trajectories. Our findings unveiled the widespread presence of both 5S rDNA and Cassandra elements in these species with low fractions (0.011%-0.110%). Notably, the Cassandra long terminal repeats (LTRs) encompass a 5S-derived domain featuring a well conserved RNA polymerase (Pol) III promoter (75.60%-80.50%), implying modular evolution with strong preservation of 5S-domain. However, the phylogenetic analysis of Cassandra 5S-derived domains revealed their formation of a distinct clade separate from 5S rDNA, and they produce RNAs with distinct secondary structures, suggesting their potential genetic divergence in these species. Furthermore, fluorescence in situ hybridization (FISH) experiments uncovered distinct chromosomal distribution patterns, with 5S rDNA localized to a specific chromosome and Cassandra elements exhibiting dispersed distribution across all chromosomes, with aggregated occurrences in subtelomeric or pericentromeric regions, emphasizing their differential evolutionary dynamics in these species. Finally, we discovered the tandem arrangement pattern of 5S rDNA and Cassandra elements in a diploid Erianthus rufipilus. Conclusions These results significantly contribute to our understanding of the sequence, abundance, structure, organization, and genomic distribution of 5S rDNA and 5S-derived Cassandra elements in sugarcane and its close relatives. Our findings indicate divergent genomic landscapes and evolutionary trajectories of 5S rDNA and 5S-derived Cassandra elements in the context of complex genomes from these species.

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