Haplotype-phased assemblies of the two poplar rust fungi species: Melampsora larici-populina and Melampsora allii-populina.

Dikaryotic rust fungi maintain two distinct haploid nuclei for most of their life cycle, making their large, repeat-rich genomes difficult to assemble and phase. Here we present haplotype-phased, near chromosome-scale genome assemblies for the poplar rust pathogens Melampsora larici-populina 98AG31 and Melampsora allii-populina 12AY07, generated using PacBio HiFi sequencing and Hi-C-guided scaffolding. For each species, we resolved 18 chromosomes per haplotype, providing the first near chromosome-level representations of poplar rust fungal species. M. larici-populina diploid assembly spans ∼203 Mb, while M. allii-populina reaches ∼416 Mb, with high completeness and strong collinearity between haplotypes. Compared with previous fragmented or collapsed references, these assemblies greatly improve contiguity, recover centromeric and telomeric features, and support the transposable element-driven genome size expansion in M. allii-populina. The haplotype-aware annotations of genes and predicted effectors derived from these resources will enable detailed analyses of genome architecture, repeat dynamics, and key loci such as avirulence genes. Together, these assemblies provide a robust genomic resource for investigating host adaptation, virulence evolution, and population diversity in poplar rust fungi.