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

Abstract 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.