Polyploidization has been recognized as a major force in plant evolution. With the continuous progress in sequencing technologies and genome assembly algorithms, high-quality chromosome-level assemblies of polyploid genomes have become increasingly attainable. However, accurately delineating these assemblies into subgenomes remains a challenging task, especially in cases where known diploid ancestors are absent. In this study, we introduce a novel approach that leverages long terminal repeat retrotransposons (LTR-RTs) coupled with the serial similarity matrix (SSM) method to assign genome assemblies to subgenomes, particularly beneficial for those without known diploid progenitor genomes. The SSM method helps identify subgenome-specific LTRs and facilitates the inference of the timing of allopolyploidization events. We validated the efficacy of the SSM approach using well-studied allopolyploid genomes, Eragrostis tef and Gossypium hirsutum, alongside artificially created allotetraploid genomes, GarGra and GmaGso. Our results demonstrated the robustness of the method and its effectiveness in assigning chromosomes to subgenomes. We then applied the SSM method to the octoploid strawberry genome. Our analysis revealed three allopolyploidization events in the evolutionary trajectory of the octoploid strawberry genome, shedding light on the evolutionary process of the origin of the octoploid strawberry genome and enhancing our understanding of allopolyploidization in this complex species.

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