G3

Fungal diseases cause devastating crop infestations and millions of deaths annually. To improve disease treatment development, we must understand the biological processes these treatments target and the fungal cellular responses they elicit. Current methods to identify genetic responses of fungi to antifungal drugs and fungicides typically involve laborious molecular genetics experiments or transcriptomics. We utilized high-throughput screens in the basidiomycete yeast Rhodotorula (Rhodosporidium) toruloides to rapidly identify genes associated with the fungal response to a copper-zinc fungicide developed by VM Agritech and compared identified genes to those transcriptionally activated in response to the fungicide in R. toruloides and the filamentous ascomycete Neurospora crassa. These 'omics technologies combined with molecular genetics and biochemical analyses showed copper and zinc synergize to inhibit fungal growth due, at least in part, to an increase in cellular zinc concentrations when copper is present. Intriguingly, although we identified genes involved in the copper-zinc fungicide response using both high-throughput screens and transcriptomics, there was little overlap in genes identified through the two methods. Our findings demonstrate the power of combining functional genomics and transcriptomics approaches in identifying a more comprehensive map of the genetic mechanisms necessary for responding to fungicides.

Biofilm formation by the fungal pathogen Candida albicans is a major source of infection. The biofilm regulator Ume6 forms complexes with partner transcription factors Efg1, Ndt80, and Upc2 to drive expression of biofilm-related genes. Here we present chromatin immunoprecipitation with sequencing (ChIP-seq) data for Ume6-chromatin association in both wild-type and efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} backgrounds. Ume6 associates with two promoter region classes. For one class, Ume6 association is significantly decreased in the efg1{Delta}/{Delta} ndt80{Delta}/{Delta} upc2{Delta}/{Delta} background. This class includes 577 genes, many with well-established roles in biofilm formation or the related process of filamentation. For the second class, Ume6 association is unperturbed in the triple mutant background. This class includes 534 genes, some with roles in iron homeostasis (e.g., SFU1) and ergosterol synthesis (e.g., ERG11), which may contribute to the impact of Ume6 on ndt80{Delta}/{Delta} mutant azole drug sensitivity. It also includes FLO9, a putative adhesin gene that is required for the emergent biofilm state produced by efg1{Delta}/{Delta} ndt80{Delta}/{Delta} double mutants. The data show that Ume6 binding to ~half of its targets requires known partners, and Ume6 binding to ~half of its targets does not. Ume6 may bind to the latter set of promoter regions independently of any partner, or perhaps with additional partners that have yet to be discovered.

Introduction. Histoplasma capsulatum is an important fungal pathogen of humans, especially in immunocompromised patients, and the World Health Organisation has listed Histoplasma as a High Priority fungal pathogen. The taxonomy of the genus Histoplasma is still under development, but its only species H. capsulatum has at least of seven lineages, and a range of sublineages. Gap Statement. Allelic multilocus sequence typing (MLST) has been very successfully applied for typing and epidemiology of pathogenic bacteria, but its adoption for fungal pathogens is more limited. Genome sequence-based MLST approaches promise much a higher resolution for comparative phylogenetics, but there are only a small number of genome assemblies available for H. capsulatum. Aim. To develop and validate genome-based MLST typing for H. capsulatum. Methodology. A total of 406 genome assemblies were generated from publicly available Illumina short-read sequencing samples. Selected genome assemblies were subsequently used to generate MLST schemes with chewBBACA, based on whole genome sequences (wgMLST), NCBI-obtained annotations (annMLST) and annotations obtained with Funannotate (fanMLST), and used for lineage identification, and comparative analyses of population structure. Results. Genome assemblies were successfully generated for 406 H. capsulatum samples, with BUSCO completeness scores of over 98%. A subset of 60 genome assemblies was used to generate and validate MLST schemes based on the 60 genome assemblies, 5 genome annotations obtained from Genbank, and 13 genome annotations generated using Funannotate, resulting in schemes with > 8,000 markers. Each of the MLST schemes allowed reproducible separation of all 7 lineages and 6 H. capsulatum Suramericanum sublineages, without the need for high-performance clusters or powerful computing equipment. Comparison of the different schemes using tanglegrams and scatter plots showed good reproducibility (r2 values of 0.8-1.0). Comparison of total sizes of genome assemblies showed significant lineage-dependent size differences, with the Suramericanum, LAmB, India, Capsulatum and Mississippiense lineages averaging a total genome size of 30-33 million bp, and the Africa and Ohiense lineages averaging at 39 million bp. Conclusion. Genome sequence- and especially genome annotation-based MLST analyses are a promising approach that can be adapted as a rapid, high-throughput method for genomic clustering and epidemiological tracing of pathogenic fungi.

A complete reference genome for species with chromosomally-determined separate sexes should contain scaffolds for all sex chromosome homologs. However, sex chromosomes present distinct computational challenges compared to autosomes. Here we present a k-mer based analysis that utilizes whole-genome sequencing of a few sex-identified isolates: Cytogenetics-By-Sequencing (CBS) tools. Unlike other approaches that typically address one aspect of the sex chromosomes, CBS-tools strives to guide users from the discovery of the heterogametic sex through identifying the sex-determination region (SDR). The core of CBS-tools is automated quantification of sex-specific k-mers in order to predict the heterogametic sex. Using publicly-available datasets, CBS-tools correctly identified the known sex-system of the 31 species tested. Additionally, we used these k-mers to verify and correct phasing of sex chromosomes between haplotypes in species representing different sex-systems. Finally, we used these k-mers to delimit the SDR boundary using an interactive web platform. CBS-tools was developed with previously unexplored sex chromosome systems in mind, but is also suitable for well-examined sex chromosome pairs.

Genetic rearrangement in the absence of selection is poorly understood. Long-read, high-depth sequencing has enabled analysis of the genetic landscape of spontaneous structural variants. We have applied PacBio Revio sequencing to identify the major classes of genetic alterations during long-term vegetative growth in Aspergillus flavus. These classes include a) precise deletions frequently associated with the flanking region of homology, b) clusters of short deletions that are heavily enriched within centromeric and subtelomeric regions that are typically 94% A+T, and c) deletions and expansions within a subset of homopolymer tracts. We find 70 homopolymer tracts longer than 44 bp, dispersed throughout the genome in noncoding regions and unassociated with transposable elements. A broader analysis of third-generation sequenced datasets from fungi found within the Phylum Ascomycota showed a high but variable prevalence of homopolymeric tracts across species. The adventitious identification of a putative flbA mutation, responsible for an altered conidial phenotype, by the Deep Variant AI system also demonstrates the power of this approach. These studies show that high-depth, long-read sequencing can probe SNPs, indels, homopolymeric tracts, and large-scale rearrangements in an unbiased fashion, and demonstrate that short deletions and homopolymer tract expansions arise spontaneously in A. flavus and other filamentous fungi.

In common wheat (Triticum aestivum L.), variation at the Vrn1 and Ppd1 loci changes plant phenology, resulting in differential adaptation useful to breeders. Because the effects of phenology on grain yield are conditional on environmental factors, the incorporation of markers for Vrn1 and Ppd1 alleles into breeding approaches has proven difficult. Historic phenotypic, genotypic, and environmental data can provide insight into the relationship between major phenology alleles and grain yield as mediated by environmental variables. Analyses of eight years of breeding trials (1,038 lines at 219 site-years) determined that weak vrn1 alleles and Ppd1 insensitivity alleles have variable effects. These effects change depending on an environment's winter temperature and latitude, respectively, but the environmentally-driven changes in effect of vrn1 alleles are much more variable than effects of Ppd1 alleles. Mediation analyses showed phenologically-dependent environmental variables are one mechanism through which phenological changes created by vrn1 and Ppd1 variants alter yield across multiple developmental stages. Environmentally-driven relationships between flowering time and yield were modeled to estimate yield allele effects as a function of effects on phenology and environmental variables. Weak winter alleles at vrn1 may play a stabilizing role on yield, due to the correlation of winter conditions that drive larger effects with later-season warm temperatures that penalize later maturity. A better mechanistic understanding of how these loci drive environment-specific yield effects may facilitate utilization of markers for these alleles and improve predictive modeling of yield.

Polyandry, multiple mating by females, both benefits females by increases genetic variation within families and provides an opportunity for sexual selection through sperm competition and cryptic female choice. Estimating polyandry in species with large clutches is challenging because genotyping all progeny individually can be impractical. We present a likelihood-based framework for estimating the number of sires and their relative contributions from the maternal genotype and pooled sequencing of entire progeny pools. The method evaluates the probability of observed pooled offspring read counts under alternative paternal genotype mixtures, accounting for Mendelian segregation and sampling variation from finite sequencing depth. We implement this framework for both biallelic SNPs and short-read mini-haplotype markers constructed from closely linked SNPs, which provide multi-allelic information comparable to traditional microsatellite markers. Simulations show that this approach accurately recovers sire number and sire contributions across a range of conditions. We then applied the method to 275 wild-caught Drosophila melanogaster females and their offspring from a Kansas population using 3,053 biallelic SNPs and 239 four-allele mini-haplotype markers. Estimates from the two marker sets were highly concordant, and combined analyses indicate that 57% of families were sired by a single male. These results demonstrate that pooled progeny sequencing provides an efficient genomic approach for estimating polyandry in species with large families.

The hybrid grouper 'Yushuban' (Cromileptes altivelis [female] x Epinephelus fuscoguttatus [male]) has shown promising performance in aquaculture trials, yet no complete reference genome has been available for this hybrid. We generated two gapless, telomere-to-telomere (T2T) haplotype-resolved genome assemblies of a single individual by integrating short-read, PacBio HiFi, Oxford Nanopore ultralong-read and Hi-C data. The maternal (Yushu_Calti_1.0) and paternal (Yushu_Efusc_1.0) haplotypes are 1,077.33 Mb and 1,083.36 Mb in length, with N50 values of 45.46 Mb and 45.55 Mb, respectively. Across all platforms, over 99.4% of reads mapped to each assembly, and Merqury-based quality values reached 57.7413 and 59.2068 (PacBio). BUSCO recovered 99.78% and 99.75% of complete core genes. We predicted 27,248 and 27,566 protein-coding genes, of which 98.52% and 99.23% were functionally annotated, and annotated telomeric motifs and repetitive elements (46.12% and 47.18% of each genome). These assemblies fill a gap in grouper genomic resources and provide a foundation for genetic dissection, parental genome comparison and molecular breeding of this hybrid.

Cell differentiation into distinct cell types generates functional specialization in eukaryotic organisms. Many fungal species undergo filamentous growth, where cells differentiate into elongated and adhesive filaments capable of expansion and invasion into new environments. The budding yeast Saccharomyces cerevisiae also undergoes filamentous growth, and the regulatory pathways that control the response have been well characterized; however, the roles essential proteins play in this response have not been systematically explored. To address this gap in understanding, we constructed a collection of 332 temperature-sensitive (ts) alleles in 320 essential genes in a strain background that undergoes filamentous growth (called {Sigma}1278b or Sigma). The ts Sigma collection showed differences in temperature sensitivity compared to a ts laboratory strain collection, revealing unexpected phenotypic diversity in essential alleles across populations of individuals. Screening the ts Sigma collection for phenotypes related to filamentous growth uncovered new phenotypes for >35% of essential alleles. New regulators of the Mitogen-Activated Protein Kinase (MAPK) pathway that regulates filamentous growth (fMAPK) were identified, including Las17, a homolog of Wiskott-Aldrich Syndrome Protein (WASP) in humans. Las17 regulated the fMAPK pathway by promoting delivery of the sensor protein, Sho1, and Rho GTPase Cdc42 to the plasma membrane. Las17 also functioned as a hub coordinating separate parallel aspects of the filamentation response. The widespread roles for essential proteins in regulating a eukaryotic differentiation response suggest broader roles essential proteins than is currently appreciated.

The allorecognition system of the hydroid Hydractinia symbiolongicarpus plays an essential role in mediating self vs. non-self recognition among cnidarian colonies, analogous to the role of the major histocompatibility complex (MHC) in bilaterian immune systems. Both complexes have contiguous stretches of similar, highly duplicated genes, suggesting a homologous relationship. Protein structural prediction methods such as AlphaFold2 also indicate structural similarity between the proteins encoded within the Hydractinia ARC and the MHC. We have determined the structure of the ARC in the related hydrozoan Podocoryna americana to determine the degree of conservation of the cnidarian allorecognition complex. Protein structural prediction methods have revealed a number of candidate allorecognition proteins that form a long contiguous complex. Phylogenies suggest homology between cnidarian allorecognition genes, as well as syntenic conservation of this gene complex across hydrozoans. Ongoing comparative genomic analyses across bilaterian species aim to clarify the relationship between the ARC and the MHC, thereby informing our understanding of the evolutionary origins of bilaterian adaptive immunity.

Genomic structural variants (SVs) are important sources of genetic diversity and contribute to the adaptation and evolution of plant-pathogenic fungi. Colletotrichum graminicola, the causal agent of maize (Zea mays L.) anthracnose, causes significant yield losses and serves as an important model for genetic studies. To investigate genomic plasticity in this pathogen, long-read genome assemblies were generated for four C. graminicola field strains collected in France, Croatia, Brazil, and Argentina and compared with the chromosome-level reference genome from the USA. Comparative analyses revealed extensive structural variation across core chromosomes, including large-scale translocations, inversions, duplications, and insertions and deletions, whereas minichromosomes showed markedly reduced collinearity among strains. Secreted proteins and predicted effectors were located significantly closer to regions affected by SVs. Approximately 174 kb of segmental duplications lacking genes were shared between core chromosome 04 and minichromosomes 11 and 13 in the reference genome but were absent from the other field strains, revealing a strain-specific structural link between core and minichromosomes. Protein clustering identified 733 candidate strain-specific genes across the reference genome. Approximately 72% of the genes annotated on the minichromosomes were strain-specific, indicating that most of their annotated genes lacked orthologous gene models in the other strains. Of the 733 candidate strain-specific genes, 492 retained homologous genomic sequences in at least one other strain and many also showed evidence of gene disruption. This pattern is consistent with lineage-specific pseudogenization and suggests that apparent gene presence-absence variation may arise through several evolutionary processes, including gene gain, gene loss and loss of gene function. Insertions and deletions in potentially regulatory flanking regions were associated with plant-pathogen interaction genes. In addition, interpretable machine learning analyses revealed a co-occurrence of duplications and LTR retrotransposons in genomic regions associated with secreted genes. This study shows that structural variation, transposable elements, and lineage-specific pseudogenization are important features of genome evolution in C. graminicola and identifies associations between genomic variation and genes involved in plant-pathogen interactions.

Premise:: Conifers comprise one-sixth of global forests and landscape genomic studies are urgently needed to understand their biology, yet relatively few conifer species have reference genomes. Targeted exon-capture is a practical alternative to whole-genome sequencing, but selecting loci without a reference genome remains a barrier. We developed a multi-tissue reference transcriptome for pi non pine (Pinus edulis) to ask how multi-tissue sampling improves probe design, and what is lost under a conventional single-tissue approach. Methods: We assembled a reference transcriptome from four diploid tissues (needle, phloem, root, vegetative bud) and one haploid tissue (megagametophyte), using the latter as a pseudo-guide. We designed a target-capture probe-set by prioritizing highly expressed P. edulis transcripts and transcripts matching a conifer exon database. Results: We recovered 76% of conserved plant genes with few duplicated transcripts. Filtering yielded 15,664 transcripts totaling 31.4 Mb, ~0.1% of the genome. Tissue-specific expression was substantial: 27.4% of transcripts occurred exclusively in non-needle tissues. Functional annotation confirmed complementary contributions, including seed-desiccation and germination genes from megagametophytes, and defense- and stress-associated genes from roots and phloem. Discussion: We built a transcriptome and efficient probe set using a roadmap suitable for other non-model plants with large genomes; keys to success were prioritizing complementary tissues and using expression-based filtering.

Breedbase ( is a comprehensive, web-based, open-source breeding management and analysis software for plant breeding that was introduced to address gaps with existing software for plant breeding data. Breedbase is continually improved and developed, with new features being added as required by the breeding community. Since its inception, the capabilities of Breedbase were improved to better serve the diverse data modeling needs of additional crops beyond our original ensemble of cassava, banana, yam, sweet potato, tomato, rice, and wheat. We describe here major updates that have been made in the past 4 years, including (1) improved handling of repetitive measurements; (2) a redesigned experimental treatments interface; (3) a more fine-grained user permission system; (4) improved trial quality control workflows; (5) intercropping support; (6) the addition of a decision meeting feature that helps decision makers make the best selection with the available data; (7) transgenics and tracking tools and finally, (8) an improved field layout tool which consolidates several trials in one field. These new features integrate seamlessly with the previous feature set of Breedbase - making it one of the most feature rich breeding management softwares available.

Dominance is an important and evolvable property of the genotype-phenotype map. A longstanding debate in evolutionary genetics concerns whether dominance is determined primarily by intrinsic properties of alleles at the focal locus or by modifiers elsewhere in the genome. Theoretical models show that second-site modifiers of dominance can evolve under some conditions. However, direct empirical tests of these models are rare because they require holding the focal heterozygous genotype constant while changing the rest of the genome. In the Drosophila montium species subgroup, female pigmentation is a Mendelian trait controlled by a single gene, POU domain motif 3 (pdm3), but the dominance relationships between pdm3 alleles differ among species. Using reciprocal introgressions among three closely related species, we compared females carrying the same Light/Dark pdm3 allele combinations in different homozygous or heterozygous genomic backgrounds and across different temperatures. In both species-pair comparisons, changing genomic background significantly altered the phenotype of identical pdm3 heterozygotes, demonstrating the existence of second-site modifiers of dominance. The effects of dominance modifiers depend on the particular pair of pdm3 alleles and on rearing temperature. These results show that dominance in this system is a genotype-by-genotype-by-environment trait, rather than a fixed attribute of individual alleles. The finding that second-site modifiers can alter dominance by reshaping reaction norms provides a developmental mechanism that links the evolution of dominance to phenotypic plasticity.

Gene duplication is an important evolutionary mechanism contributing to genomic innovation and adaptation. While the long term fate of duplicated genes has been extensively studied, immediate effects such as those resulting from increase in gene dosage remain understudied. Of particular interest is the question of how expression level relates to the optimum after a duplication, as it could fall short, exceed or directly reach the optimal expression. In this study, we investigated the relationship between expression and fitness for 11 genes previously reported to increase fitness when duplicated in Saccharomyces cerevisiae to compare the expression level achieved by duplication to the one maximizing fitness. While most of the studied genes show little fitness variation across the expression levels tested, we found that ESP1, which encodes a separase essential to anaphase initiation, exhibits a clear fitness optimum at an expression level above the native single-copy state. However, the expression level associated with gene duplication exceeds the optimum. We find that ESP1 overexpression is associated with a reduced cell size and a lower proportion of the cell cycle spent in G2 phase, consistent with its role in cell cycle progression. We propose that the duplication of ESP1 accelerates the cell cycle, resulting in smaller, faster-dividing cells. When measured in different environments, the fitness functions also exhibit similar shapes, with slight variation in the optimal expression. Our findings highlight how gene duplication can confer an immediate selective advantage through dosage increase while being non-optimal, reversing the directionality of natural selection on expression level after duplication.

Epigenetic information is required for the maintenance of gene expression states through cell division. Some epigenetic information can also persist through generations, raising the possibility that epigenetic differences may arise in populations and contribute to evolutionary processes such as drift and natural selection. Evaluating this requires characterising the properties of such epimutations, so that their rate, stability and genome-wide distribution can be assessed. Recent experiments in many organisms have demonstrated that epimutations occur, but have limited stability relative to DNA sequence mutations. However, the extent to which epimutations contribute to evolution is still unclear, in part because it is not clear whether epimutations respond dynamically to environmental conditions. Here, we use the fission yeast S. pombe, in which evolutionarily relevant epimutations have been observed, but not yet systematically characterised, to test whether a physiologically relevant stress, ethanol exposure, leads to changes in the properties of epimutations. Using laboratory evolution experiments with repeated population bottlenecks to minimise selection, we show that epimutations associated with changes in small interfering (si)RNAs occur at a greater rate in ethanol. Ethanol-induced epimutations are enriched at transposable element loci and correlate with changes in transposon expression. Ethanol exposure also increases gene expression noise genome-wide and variability in growth rate; importantly, both these increases are suppressed in mutants lacking siRNAs. Together, these results demonstrate that stress can alter the generation of heritable epigenetic information, and suggest that siRNAs may have a role in promoting phenotypic variability in response to environmental stress.

Generating high-quality genome assemblies for small animals with large genomes is complex due to their small body size, DNA contamination, and repetitive elements. Ticks exemplify these complexities, while also being a global health threat to humans, domestic animals, and wildlife. Advances in long-read sequencing platforms now make it feasible to obtain large amounts of raw sequence data from individual specimens, but challenges remain. Key genome workflow challenges include error correction, assembly, transposable element annotation, and chromosome assignment and synteny. Here we examine three individual Dermacentor reticulatus ticks using deep Oxford Nanopore sequencing. Comparing and contrasting bioinformatic tools for raw read and assembly manipulation allows us to identify the parameters that provide a high-quality haploid genome assembly among 38 assemblies. We find Dorado error corrected raw read data from approximately two flow cells are needed, but limiting assembly coverage to 40x produces the highest quality assemblies. Then we examined the effects of optimal workflow parameters on downstream analyses of gene synteny and manual transposable element annotation of the three tick assemblies in comparison to an independent assembly of a fourth D. reticulatus individual from a different country. Gene synteny analysis allows chromosome assignment of scaffolds and transposable element identification was improved markedly with manual curation. Finally, we compared the genetic variation of D. reticulatus across two populations and found similar genetic diversity. Our study provides a clear workflow to obtain high-quality assemblies from Oxford Nanopore sequences and genetic characterization of a species with a large, complex, and repetitive genome.

Plant pathogen genomes adapt across coding and non-coding regions in response to their hosts, environments, and disease management practices. Venturia inaequalis, the causal agent of apple scab, has evolved alongside its Malus host throughout apple domestication. We generated 17 high-quality genome assemblies, including the first chromosome-scale assembly of V. inaequalis, to define the species' complete gene content and investigate the role of structural variation in genome evolution. Together with a previously published scaffold-level reference, these assemblies were used to construct a gene-based pangenome containing 215,301 predicted protein-coding genes clustered into 13,240 orthogroups, 88.11% of which were core (73.86%) or soft-core (14.25%), 11.88% shell (present in 2-16 isolates), and 0.1% private, indicating a highly conserved gene repertoire. Virulence-associated effectors were significantly enriched in the soft-core and shell components, and accessory apoplastic effectors displayed elevated nucleotide diversity and nonsynonymous-to-synonymous substitution ratios (Ka/Ks) relative to core effectors, consistent with diversification. We further identified a putative accessory chromosome of variable size (23-132.6 kb) in European and U.S. isolates encoding genes with virulence function. A graph-based pangenome identified 38,799 non-redundant structural variants (SVs), largely driven by Gypsy LTR retrotransposons. We used the graph-based pangenome to genotype 136 globally distributed V. inaequalis isolates and identified variants differentiating populations associated with domesticated apple (Malus domestica) from those associated with its Central Asian wild progenitor, M. sieversii. Together, these resources establish a foundation for future studies of diversity, host adaptation, and genome evolution in V. inaequalis.

The opportunistic human pathogen Candida albicans depends on the unfolded protein response (UPR) for cell wall integrity, antifungal tolerance, filamentous growth, and virulence. The UPR is driven by the conserved transmembrane sensor Ire1, which is activated either by misfolded proteins through its luminal domain or by lipid bilayer stress (LBS) through its transmembrane domain. In budding yeast, these two activation modes deploy divergent transcriptional programs. Whether the requirement for these two input domains is separable in C. albicans, where the cell membrane and cell wall are themselves the targets of major antifungal drug classes, remains unknown. Here, we engineered a C. albicans strain expressing Ire1 lacking an intact luminal domain (ire1{Delta}LD), which no longer detects proteotoxic stress. The ire1{Delta}LD strain grew in the presence of the azole antifungals fluconazole and miconazole but was highly sensitive to heat shock, cell wall stress, and the echinocandin caspofungin. It was also unable to sustain filamentous growth and showed reduced virulence in a Caenorhabditis elegans infection model. RNA sequencing revealed only modest changes to the steady-state transcriptome of ire1{Delta}LD cells. Together, these findings define a differential requirement for the input domains of C. albicans Ire1, uncoupling growth under azole-induced membrane stress from the cell wall, thermal, and virulence-associated outputs that depend on proteotoxic sensing, a distinction that could inform antifungal strategies targeting the UPR.

Phakopsora pachyrhizi, the causal agent of soybean rust, is among the most aggressive and economically impactful pathogens affecting soybean (Glycine max) production worldwide. As a dikaryotic rust fungus, P. pachyrhizi maintains its genetic information as two discrete haplotypes that are sequestered within separate nuclei for most of its life cycle as it reproduces asexually via the production of infectious urediniospores. Previous genomes of three P. pachyrhizi isolates collected in South America revealed a large genome with a high transposable element content, measuring up to 1.25 Gb and 93%, respectively. Although these efforts provided valuable foundational resources, the complexity of the genome resulted in highly fragmented assemblies. Here, we generated a highly contiguous, substantially improved, annotated genome resource from the P. pachyrhizi isolate LA04-1, collected during the initial outbreak of soybean rust in the continental United States in 2004. The assembly is haplotype-phased, with each haplotype assembled into 18 chromosomes totaling 572 Mb and 569 Mb in size. The isolate contains unique mating-type genes and does not share haplotype genomes with any available isolates with whole-genome sequencing datasets. This resource will be valuable to future investigations into the underlying mechanisms of virulence in this important plant pathogen.