Haplotype-phased genomes of the barley leaf rust pathogen reveal evidence of repeat element expansion and somatic hybridization.

Barley leaf rust disease, caused by Puccinia hordei, leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for 10 globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed 7 distinct lineages of P. hordei, including evidence of nuclear exchange, with a shared nuclear haplotype detected between 2 US lineages. Nuclear genome sizes ranged from ∼140 to 147 Mbp, with the exception of isolate 90ISR03 from Israel (∼163 Mbp), which also harbored a 6.2-Mbp extra scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content (∼70%) than related cereal rust fungi, driven primarily by the proliferation of long terminal repeat (LTR) retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were conserved across all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the United States and globally, and nuclear exchange plays a role in generating novel diversity. Genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as an extra chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei.