Genome structural plasticity and copy number dynamics shape adaptive evolution in a barley fungal pathogen

Leaf rust, caused by Puccinia hordei , is one of the most important diseases of barley globally. Here, we report the first haplotype-resolved chromosome-level genome assembly for P. hordei . To gain deeper insight and understanding of the P. hordei genome, we generated such assemblies for two Australian isolates with contrasting virulence, one collected in the 1970s and one in 2001. The two haplotype-phased and chromosome-level assemblies were used to investigate genome architecture in P. hordei and to investigate variability and evolutionary relationships of 41 virulence-divergent isolates that were collected over a 54-year period (1966–2020) in Australia. We report, for the first time in a rust fungus, evidence of a conserved chromosome, chromosome 9, which is enriched for structural variation and accessory features that imply it functions as a compartmentalized core chromosome with regions subject to rapid diversification, potentially acting as a structural hotspot in adaptive evolution in P. hordei . Clear genetic stratification into clonal and recombining lineages was uncovered, with evidence of both long-term asexual propagation and recombination. Analyses of haplotype-resolved mating-type gene phylogenies and chromosome 9 k-mer profiles confirm divergence at key loci underpinning reproductive modes, distinguishing clonally derived from sexually recombined groups. We also identified pervasive copy number variation across the P. hordei genomes, with clonal lineage-specific duplications. Consistent with this finding, the study for the first time links copy number variation of the Cyp51 gene to fungicide insensitivity. This latter discovery is highly significant as it represents the first documented case of fungicide insensitivity in a rust pathogen in which an underlying genetic mechanism has been identified. Together, these findings illuminate how genome structure, recombination, and structural variation shape the evolution and adaptation of P. hordei , providing a guidance for future surveillance and management of this pathogen.