Genome Sequence and Population Genomic Analysis of <i>Cronartium quercuum</i> f.sp. <i>fusiforme</i>

ABSTRACT Fusiform rust, caused by Cronartium quercuum f. sp. fusiforme ( Cqf ), is the most severe disease of planted southern pines in the south‐eastern United States. Despite the critical role pathogen genomics play in disease outcomes, the population structure and genetic variability of Cqf remain poorly characterised. To address this gap, we generated a high‐quality chromosome‐scale genome assembly of Cqf and leveraged this resource to perform the population genomic analyses of 274 haploid pycniospore samples derived from controlled inoculations with geographically diverse aeciospore collections. The assembled Cqf genome (87.03 Mbp across 19 contigs) displayed high completeness (BUSCO: 91.1%) and structural continuity (Scaffold N50: 5.4 Mbp), establishing a robust reference for population‐genetic analysis. PCA, DAPC and ADMIXTURE revealed a pronounced east–west structure indicating five genetically distinct metapopulations (Western Gulf, Upper Central Gulf, Lower Central Gulf, Lower Atlantic Coast/Eastern Piedmont, Mid‐Atlantic Coast). AMOVA revealed that high levels of variation exist in Cqf , with the greatest proportion of genetic variation occurring in local populations (85.5%) with moderate differentiation among regional metapopulations ( Φ = 0.117). Sliding‐window F ST analyses highlighted the genomic regions of elevated differentiation, including loci encoding candidate secreted effector proteins. Genetic diversity metrics revealed evidence of non‐random mating in some regions. These results refine the understanding of Cqf population structure, confirm the relevance of USDA Resistance Screening Center geographic zones and provide novel genomic resources to support breeding for durable fusiform rust resistance in southern pines.