Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.

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Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with nonhaploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the 2 nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements 2 previously released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these 3 Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with non-haploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the two nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements two previous released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) that was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully-phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these three Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

Generation of genetic diversity is crucial for the evolution of new traits, with mutation and sexual 34 recombination as the main drivers of diversity in most eukaryotes. However, many species in the 35 fungal kingdom can propagate asexually for extended periods and therefore understanding alternative 36 mechanisms contributing to genetic diversity in asexual populations has been of great interest 1,2 . Some 37 fungi can use a parasexual mechanism to exchange genetic material independently of meiosis 2 . This 38 process involves anastomosis of haploid hyphae and fusion of two nuclei to generate a single diploid 39 nucleus, which subsequently undergoes progressive chromosome loss to generate recombinant haploid 40 offspring. Parasexuality has been described in members of the ascomycete phylum (64% of described 41 fungal species) in which the dominant asexually propagating form is haploid 3 . However, in 42 basidiomycete fungi (34% of described species), the predominant life stage is generally dikaryotic, 43 with two different haploid nuclei maintained within each individual 3 . The role of non-sexual genetic 44 exchange between such dikaryons in generating genetic diversity is not known. 45 Basidiomycetes include many fungi with critical ecosystem functions, such as wood decay and 46 plant symbiosis, as well as agents of important human and plant diseases 1 . Rust fungi (subphylum 47 Pucciniomycotina) comprise over 8,000 species including many pathogens of major agricultural and 48 ecological significance 4 . These organisms are obligate parasites with complex life cycles that can 49 include indefinite asexual reproduction through infectious dikaryotic urediniospores. Early researchers 50 speculated that rust fungi can exchange genetic material during the asexual phase 5-8 , but these 51 hypotheses could not be confirmed molecularly. Some naturally occurring rust pathotypes have been 52 suggested to have arisen by somatic hybridisation and genetic exchange based on limited molecular 53 evidence of shared isozyme or random amplified polymorphic DNA (RAPD) markers 9,10 . Mechanisms 54 underlying genetic exchange are unknown, but may involve hyphal anastomosis followed by nuclear 55 exchange and/or nuclear fusion and recombination 11 . Recent advances in assembling complete karyon 56 sequences in rust fungi 12,13 provide the opportunity to definitively detect and discriminate between 57 nuclear exchange and recombination. 58 4The Ug99 strain (race TTKSK) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici 59 (Pgt) presents a significant threat to global wheat production 14 . It was first detected in Uganda in 1998 60 and described in 1999 15 , and has since given rise to an asexual lineage that has spread through Africa 61 and the Middle East causing devastating epidemics 14 . The origin of the Ug99 lineage is unknown, 62 although it is genetically distinct from other Pgt races 16,17 . To resolve the genetic makeup of Ug99, we 63 generated a haplotype-phased genome reference for the origi...

Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.