Ancestral Karyotype Reconstruction Reveals Chromosome Rearrangements and Large Inversions in Oryza

A stable chromosome count does not imply a static genome. Oryza species have largely retained a basic chromosome number of 12, yet most rearrangements are described against extant references, making ancestral and derived orientations difficult to distinguish. We reconstructed an ancestral Oryza karyotype (AOK) containing 12 chromosomes and 23,010 ordered genes from 24 chromosome-level genome assemblies representing 34 ingroup genome/subgenome units. Unlike a modern reference, the AOK provides gene-order polarity, allowing rearrangements detected in different species and population panels to be compared in the same evolutionary direction. AOK-polarized analysis of 400 rice assemblies revealed structural change at two scales. Across species, the derived allotetraploid karyotypes of Oryza alta and Oryza grandiglumis were parsimoniously accounted for by three reciprocal translocations. Within rice populations, four principal inversion-associated loci showed contrasting lineage distributions. The most complex locus, on chromosome 6, comprised nested structural haplotypes that were unevenly distributed among rice populations and associated with published variation in seedling cold tolerance. These findings show how lineage-specific chromosome exchanges and polymorphic inversion complexes have reshaped a numerically conserved karyotype. More broadly, an ancestral gene-order reference links deep karyotype evolution with standing structural diversity and provides a framework for tracing the direction of chromosome rearrangements during rice diversification.