Haplotype-phased and chromosome-level assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust

Rust fungi are characterized by large genomes with high repeat content, and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we describe a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently the largest fungal genome assembled to chromosome scale. Transcript-based annotation identified 47,512 genes with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat content, with genome-size expansion, gene losses and gene family expansions suggested by multiple copies of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species with smaller genomes. Fine-time-point transcriptomics revealed seven clusters of co-expressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a “two-speed genome” evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population drifted from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing evolution and population differentiation. The high-quality assembly provides valuable genomic resources for future studies on the evolution of P. polysora.