Rita Tam1, Mareike Möller1, Runpeng Luo1, Zhenyan Luo1, Ashley Jones1, Sambasivam Periyannan1,2,3, John P. Rathjen1 and Benjamin Schwessinger1 1Research School of Biology, Australian National University, Canberra ACT 2601, Australia; 2Commonwealth Scientific and Industrial Research Organisation Agriculture and Food, Canberra ACT 2601, Australia; 3School of Agriculture and Environmental Science, Centre for Crop Health, University of Southern Queensland, Toowoomba, Queensland 4350, Australia Corresponding authors: john.rathjenanu.edu.au, benjamin.schwessingeranu.edu.au Abstract

Phased telomere-to-telomere (T2T) genome assemblies are revolutionizing our understanding of long-hidden genome biology “dark matter” such as centromeres, rDNA repeats, inter-haplotype variation, and allele-specific expression (ASE), yet insights into dikaryotic fungi that separate their haploid genomes into distinct nuclei are limited. Here, we explore the impact of dikaryotism on the genome biology of a long-term asexual clone of the wheat pathogenic fungus Puccinia striiformis f. sp. tritici. We use Oxford Nanopore Technologies (ONT) duplex sequencing combined with Hi-C to generate a T2T nuclear-phased assembly with >99.999% consensus accuracy. We show that this fungus has large regional centromeres enriched in LTR retrotransposons, with a single centromeric dip in methylation that suggests one kinetochore attachment site per chromosome. The centromeres of homologous chromosomes are most often highly diverse in sequence, and kinetochore attachment sites are not always positionally conserved. Each nucleus carries a unique array of rDNAs with more than 200 copies that harbor nucleus-specific sequence variations. The inter-haplotype diversity between the two nuclear genomes is shaped by large-scale structural variations linked to transposable elements. ONT long-read cDNA analysis across dormancy and distinct host infection conditions revealed pervasive ASE for ∼20% of the heterozygous genes. Genes encoding secreted proteins, including putative virulence effectors, are significantly enriched in ASE genes that appear to be linked to elevated CpG gene body methylation of the lower-expressed allele. This suggests that epigenetically regulated ASE is likely a previously overlooked mechanism facilitating plant infection. Overall, our study reveals how dikaryotism uniquely shapes key eukaryotic genome features.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.280359.124.

Freely available online through the Genome Research Open Access option.

Received December 12, 2024. Accepted April 11, 2025.