Haplotype-specific chromosome painting unveils recombination patterns in the holocentric species Rhynchospora breviuscula H.Pfeiff

The genus Rhynchospora Vahl (beak-sedges) comprises approximately 381 accepted species with a worldwide distribution, all of which possess holocentric chromosomes, where centromeric activity is distributed almost along the entire chromosome. Despite the recent advances, the mechanisms governing the dynamics of meiotic recombination in holocentric plants remain poorly understood. Here, we developed haplotype-specific oligo-FISH probes for chromosomes 1, 2, and 3 based on a haplotype-phased genome assembly of Rhynchospora breviuscula ( n = 5), enabling homolog-specific chromosome painting. Each probe set was labelled with a distinct fluorophore and hybridised in situ to metaphase chromosomes of the reference plant and seven F ₁ individuals derived from self-crossed reference plants. This approach allowed the unambiguous discrimination of homologous haplotypes and the indirect visualisation of crossover (CO) events in recombined chromosomes. We observed that recombination events were predominantly located in terminal chromosomal regions, consistent across individuals. These results corroborate previous findings from single-cell recombination mapping and provide independent cytological validation of the recombination landscape in this species. Our study establishes haplotype-specific chromosome painting as a robust tool for high-resolution mapping of meiotic recombination in holocentric plants across generations. Furthermore, these probes provided a foundation for future investigations into inverted meiosis, a mechanism characterized by an alternative pattern of chromosome segregation in holocentric species.