Water caltrop (Trapa spp., Lythraceae) is a traditional but currently underutilized non-cereal crop. Here, we generated chromosome-level genome assemblies for the two diploid progenitors of allotetraploid Trapa. natans (4x, AABB), i.e., diploid T. natans (2x, AA) and Trapa incisa (2x, BB). In conjunction with four published (sub)genomes of Trapa, we used gene-based and graph-based pangenomic approaches and a pangenomic transposable element (TE) library to develop Trapa genomic resources. The pangenome displayed substantial gene-content variation with dispensable and private gene clusters occupying a large proportion (51.95%) of the total cluster sets in the six (sub)genomes. Genotyping of presence-absence variation (PAVs) identified 40 453 PAVs associated with 2570 genes specific to A- or B-lineages, of which 1428 were differentially expressed, and were enriched in organ development process, organic substance metabolic process and response to stimulus. Comparative genome analyses showed that the allotetraploid T. natans underwent asymmetric subgenome divergence, with the B-subgenome being more dominant than the A-subgenome. Multiple factors, including PAVs, asymmetrical amplification of TEs, homeologous exchanges (HEs), and homeolog expression divergence, together affected genome evolution after polyploidization. Overall, this study sheds lights on the genome architecture and evolution of Trapa, and facilitates its functional genomic studies and breeding program.

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