Reference-free identification and pangenome analysis of accessory chromosomes in a major fungal plant pathogen

Accessory chromosomes, found in some but not all individuals of a species, play an important role in pathogenicity and host specificity in fungal plant pathogens. However, their variability complicates reference-based analysis, especially when these chromosomes are missing in the reference genome. Pangenome variation graphs offer a reference-free alternative for studying these chromosomes. Here, we constructed a pangenome variation graph for 73 diverse <i>Fusarium oxysporum</i> genomes, a major fungal plant pathogen with a compartmentalized genome that includes conserved core as well as variable accessory chromosomes. To obtain insights into accessory chromosome dynamics, we first constructed a chromosome similarity network using all-vs-all similarity mapping. We identified eleven core chromosomes conserved across all strains and a substantial number of highly variable accessory chromosomes. Some of these accessory chromosomes are host-specific and likely play a role in determining host range. Using a k-mer based approach, we further identified the presence of these accessory chromosomes in all available (581) <i>F. oxysporum</i> assemblies and corroborated the occurrence of host-specific accessory chromosomes. To further analyze the evolution of chromosomes in <i>F. oxysporum</i>, we constructed a pangenome variation graph per group of homologous chromosomes. This reveals that accessory chromosomes are composed of different stretches of accessory regions, and possibly rearrangements between accessory regions gave rise to these mosaic accessory chromosomes. Furthermore, we show that accessory chromosomes are likely horizontally transferred in natural populations. Our findings demonstrate that a pangenome variation graph is a powerful approach to elucidate the evolutionary dynamics of accessory chromosomes in <i>F. oxysporum</i>, which is not only a useful resource for <i>Fusarium</i> but also provides a framework for similar analyses in other species containing accessory chromosomes.