A chromosome-scale, haplotype-resolved genome assembly of the barley stripe rust pathogen Puccinia striiformis f. sp. hordei

Puccinia striiformis f. sp. hordei (Psh) causes barley stripe rust, an economically important disease affecting barley production across multiple temperate regions. Unlike its well-studied wheat-infecting relative P. striiformis f. sp. tritici, genomic resources for Psh remain limited, with no fully documented chromosome-scale, haplotype-resolved reference genomes publicly available. Here we present a high-quality, haplotype-resolved genome assembly of the dikaryotic Psh isolate NP85002, generated using Oxford Nanopore long-read sequencing combined with Hi-C chromatin conformation capture. The assembly comprises 18 chromosomes per haplotype (151.3 Mb total) including 33 telomere-to-telomere chromosomes, telomeric repeats at 69/72 chromosome ends, and six internal gaps. The assembly shows high consensus accuracy (QV >72) and strong haplotype separation (97.54% within-haplotype Hi-C contacts). Genome completeness reached 90.1% complete BUSCOs, and a combined lift-over plus ab initio annotation achieved 94.4% complete BUSCOs. Repeat annotation indicates 45% repetitive content. We additionally provide a 102,058 bp mitochondrial genome assembly with 40 annotated genes. This genome resource provides a chromosome-scale framework for comparative and population genomic analyses of barley stripe rust and related cereal rust pathogens.