G3

In fungi, hyphal fusion is beneficial within an individual, but fusion between individuals comes with the risks of infection or exploitation. To manage this risk, fungi have developed mechanisms to restrict sustained fusion to be within a genetic individual, called allorecognition. In Ascomycete fungi, this recognition is based on allelic identity at several polymorphic allorecognition genes, often triggering cell death. However, the genetic basis of allorecognition is unknown in basidiomycetes, the clade that includes mushroom-forming fungi. Here, we map the first locus for this trait, which we call somA, in the mushroom-forming fungus Coprinopsis cinerea. We combined F1 offspring phenotypes with independent backcross lines to identify a region on chromosome 5 linked with the production of a barrage zone, a classic allorecognition phenotype. Fine-mapping of this region resulted in a region with a set of kinases and NACHT domain proteins, flanked by a leucine-rich repeat (LRR) protein. While the NACHT and kinase proteins are diverse between the parents, the LRR-encoding protein shows signs of purifying selection. Additional C. cinerea genomes show that this region contains several highly divergent alleles, consistent with long-term balancing selection. These polymorphic alleles all contain a single monomorphic LRR, which may indicate a novel mechanism for fungal nonself recognition. Based on a phylogenetic survey of related Basidiomycetes, this specific locus architecture appears to be restricted to closely related species. This finding of a multiallelic locus may explain the general trend of few nonself recognition loci in basidiomycetes. These results provide a first understanding of how individuality is maintained in basidiomycetes.

Gene duplication typically fails to confer a selective advantage to an organism, prompting their removal from a population. In the rare instance that duplication either does not incur a fitness cost or it enhances fitness, gene families can form through repeating the duplication process. While the function of gene duplicates has been studied in detail, little work has explored how repeated duplication impacts paralog redundancy and may restrict the emergence of new paralogs or novel function. Here, we constructed a panel of single deletion mutants for each of the 14 members of the <em>Candida albicans</em> telomere-associated (<em>TLO</em>) gene family to test the redundancy in molecular and biological function among paralogs from a lineage-specific expansion. Tlo proteins function as interchangeable subunits of the Mediator transcriptional regulatory complex and have the potential to alter gene expression and an array of cellular responses. Redundancy was the most common outcome, being observed for approximately 80% of the phenotypic assays in strains lacking single <em>TLO </em>genes. However, mutants for all 14 paralogs displayed non-redundant functions in phenotypes ranging from carbon utilization to <em>in vivo</em> virulence. Analysis of gene expression in single <em>TLO </em>mutants found similar trends in redundancy, and loss of single <em>TLO</em>s disproportionately affected genes involved in filamentation, adhesion, redox reactions, and transporter activity at the cell surface. Importantly, sequence divergence between paralogs positively correlated with the frequency of altered phenotypes in single <em>TLO </em>mutants, indicating the acquisition of non-redundant function with increased evolutionary distance. Double mutants lacking two <em>TLO</em> genes produced both positive and negative synergistic phenotypes, suggesting that crosstalk or coordinated regulation is common among paralogs. Together, this study demonstrates that recently emergent paralogs acquire non-redundant functions despite often retaining redundancy with other gene family members to form a highly interconnected functional network.

The Agaricomycotina accounts for roughly a third of all described fungi. They are important due to their wide range of lifestyles and economic and environmental relevance. Certain agaricomycetes act as lignocellulose degraders, playing a significant role in forest ecosystems and bioremediation processes. These wood-decaying fungi have historically been classified as mostly white- or brown-rot based on their ability to degrade lignin, with white-rot fungi possessing a collection of lignocellulose-degrading enzymes, which are reduced or absent in brown-rot fungi. Here, we sequenced and annotated the genome of the agaricomycete Crepidotus cesatii CBS 511.95 and explored its genome and predicted enzymatic content in a comparative context. The 36.04 Mbp genome is in 235 scaffolds, with 3.34% repeat content and 12,891 predicted genes. We found that the PFAM distributions of identified orthogroups suggested that C. cesatii shows patterns more similar to white-rot fungi compared to brown-rot fungi. Additionally, C. cesatii contained multiple copies of CAZymes CBM1 and AA9 involved in hydrolysis of lignocellulose, similar to white-rot fungi. On the other hand, according to the Conserved Unique Peptide Patterns (CUPP) data for AA2 peroxidases, the key enzymes in lignin degradation, C. cesatii is more similar to brown-rot fungi. Based on our analyses we predict that C. cesatii is another representation of the continuum of wood decaying modes between white and brown rot fungi combining genetic features of both types of fungi.

A highly contiguous and complete reference genome of Cercospora cf. flagellaris, the causal agent of foliar disease on many plant hosts including Cercospora leaf blight of soybean, was assembled using a combination of PacBio and Illumina sequencing reads. The genome assembly is 33.72 Mb in length and consists of 14 nuclear scaffolds and one mitochondrial contig. Four scaffolds have telomeric repeats on both ends and represent fully assembled chromosomes, while nine scaffolds represent partially assembled chromosomes with telomeric repeats on one end. The assembly has an N50 of 2.90 Mb and an L50 of 5 scaffolds. Genome annotation identified 11,268 genes, of which 947 and 360 were predicted to encode secreted proteins and effectors, respectively. Additionally, 512 genes were predicted to encode carbohydrate-active enzymes and 60 biosynthetic gene clusters were annotated. Taken together, this annotated genome assembly will be a valuable resource for genomics, host-pathogen interactions, and population biology research in this economically important pathosystem.

The Candida Genome Database (CGD; www.candidagenome.org) is both a model organism database and a fungal pathogen database. As a model organism database, CGD stores data for Candida albicans, which serves as a model species both for other Candida spp. and for non-Candida fungi that form biofilms and undergo routine morphogenic switching. As a fungal pathogen database, CGD now hosts locus pages for six species of the best-studied pathogenic fungi in the Candida group. Pathogenic Candida species have become increasingly drug resistant and there is thus a pressing need for research into basic Candida biology, epidemiology, phylogeny, and potential new antifungals, as well as a single location where all of the available data are collected, curated, and made easily searchable. CGD curates the gene-based Candida experimental literature in real time, extracting, organizing and standardizing gene annotations. CGD also links clinical data on disease to relevant Literature Topics to improve searchability for clinical researchers. Because CGD curates the literature for multiple species and most research focuses on aspects related to pathogenicity, we focus our curation efforts on assigning Literature Topic tags, collecting detailed mutant phenotype data, and assigning controlled Gene Ontology terms with accompanying evidence codes. Our Summary pages for each locus include the primary name and all aliases for that locus, a description of the gene and/or gene product, detailed ortholog information with links, a synteny view, a JBrowse window with a visual view of the gene on its chromosome, links to Phenotype, Gene Ontology, Interactions, and Expression pages, as well as sequence information, references cited on the summary page itself, and any locus notes. The database also serves as a community hub, where we link to various types of reference material of relevance to Candida researchers, including colleague information, news, and notice of upcoming meetings. We routinely survey the community to learn how the field is evolving and how needs may have changed. Here we describe CGDs new modern web interface and multiple new tools that have been added in the last 6 months, allowing, among other things, users to better understand the available expression data for a locus and seamlessly switch between species for a given locus.

Gene model for the ortholog of raptor in the D. grimshawi May 2011 (Agencourt dgri_caf1/DgriCAF1) Genome Assembly (GenBank Accession: GCA_000005155.1) of Drosophila grimshawi. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the ~65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

Understanding the genomic consequences of thermal adaptation in fungi is crucial, as rising global temperatures are expected to have negative impacts on food safety and human health. The family Chaetomiaceae contains a large number of thermophilic fungal taxa, but previous studies have reported inconsistent optimal growth temperatures (OGT) for the same strains, obtained with various laboratory methods. Here we applied a standardized laboratory approach to measure OGT across strains of 17 Chaetomiaceae species and used a phylogenomic approach to test associations between OGT, rates of genome evolution, and strength of purifying selection. Compared to mesophiles, thermophilic fungi showed faster nucleotide substitution rates. In addition, thermophiles showed lower dN/dS ratios than mesophiles, suggesting stronger purifying selection on conserved orthologs. We hypothesize that the elevated substitution rates are linked to high growth rates, as thermophilic fungi grew significantly faster than mesophilic ones. Our results show that selective pressures may act at different temperatures for distinct genomic characteristics. Genome size was lower at OGT [&ge;] 35{degrees}C compared to mesophilic species, while GC content did not show a large difference between mesophiles and thermotolerant species, but increased in thermophilic species with an OGT [&ge;] 45{degrees}C.

ObjectivesModel species are essential for fundamental research in biology. While a complete genomic sequence is a prerequisite for genetic studies, it is not enough on its own. Understanding the three-dimensional organization of the genome is also important, allowing researchers to gain a more realistic understanding of the mechanisms governing genome function. In the fungal model Podospora anserina, although the genomic sequence has been established for a long time, the three-dimensional organization remained unknown. Here we obtained the first Hi-C datasets and present associated 3D models, providing the research community with a valuable resource for better multi-omics data integration. Data descriptionHi-C experiments were performed in duplicate, using nuclei purified from wild-type fungal mycelium. Four FASTQ files were obtained (two per replicate) and used as inputs for the 3DGB workflow with four different output resolutions, to observe the genome organization of P. anserina at different levels of detail (50 kb, 20 kb, 10 kb, and 5 kb). In a context where researchers already have, for this species, a large amount of traditional omics data (ChIP-seq, RNA-seq, etc.), these 3D models are helpful for complementing the linear representation of the genome, which is traditionally used in bioinformatic analyses.

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

Gene model for the ortholog of tango (tgo) in the Sep. 2015 (UC Berkeley ASM127793v1/DbusGB1) Genome Assembly (GenBank Accession: GCA_001277935.1) of Drosophila busckii. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Carpobrotus chilensis (Chilean sea fig) is a coastal succulent of uncertain origin that has naturalized along the California coast, where it co-occurs and hybridizes with the invasive species, Carpobrotus edulis. Despite their ecological importance and widely supported hybridization, genomic resources for this genus remain scarce. Here, we present a draft genome assembly of C. chilensis generated from PacBio HiFi long reads. The assembled nuclear genome spans 981.7 Mb across 178 contigs. The contig N50 was 73.0 Mb, and BUSCO completeness was 96.3%. K-mer and SNP-based analyses indicate extremely low heterozygosity (3.4 x 10-), reduced genetic diversity in this population. The genome is highly repetitive, with 81.67% of the sequences composed of transposable elements, predominantly long terminal repeat (LTR) retrotransposons. Gene prediction identified 21,744 protein-coding genes, with BUSCO completeness of 95.8%. Comparative analysis with C. edulis identified 8,783 single-copy orthologous gene pairs, with a median synonymous substitution rate (dS) of 0.019, indicating low sequence divergence between the two species. This genome assembly provides a foundational resource for investigating the genomic basis of hybridization and invasion in Carpobrotus.

Degenerate sex chromosomes (e.g., the Y or W) remain among the most difficult regions of eukaryotic genomes to assemble because they are highly repetitive and structurally complex. While many recent lepidopteran genome assemblies contain W chromosome scaffolds, the accuracy and consistency of these assemblies remain uncertain, due to lack of replication within species. However, the silkworm moth Bombyx mori is an exception, with numerous independent W chromosome assemblies currently available. We compared six independent long-read W chromosome assemblies, which proved to be highly inconsistent in structure, even among nominally identical genotypes. In contrast, autosomes and the Z chromosome were highly concordant among these assemblies, indicating that current assemblies remain unreliable for resolving W chromosome structure. Additionally, we analyzed repetitive DNA content across the genome. First, we combined assembly- and read-based repeat-discovery methods to generate a comprehensive and curated Bombyx repeat library, which we make publicly available. Assessing repeat content and diversity, we find that the W chromosome is comprised almost entirely of repetitive DNA but that the richness and divergence of W repeats are substantially reduced compared to the remainder of the genome. This reduced diversity, initially inferred from assemblies, is confirmed by direct analysis of PacBio HiFi sequencing reads partitioned by chromosome. We also demonstrate that the B. mori p50ma genome assembly (the current NCBI RefSeq assembly) carries a W chromosome and mitochondrial genome introgressed from B. mandarina. This discovery provided an opportunity to investigate patterns of divergence between closely related W haplotypes, revealing substantially more rapid turnover of repeat content on the W than elsewhere in the genome. Together, our results show that current W chromosome assemblies, although structurally flawed, nevertheless capture robust biological patterns of repeat diversity and support the hypothesis that rapid repeat turnover, rather than frequent chromosome replacement, may underlie the apparent lack of W chromosome homology across Lepidoptera.

Gene model for the ortholog of raptor in the May 2011 (Agencourt Dere_CAF1/DereCAF1) Genome Assembly (GenBank Accession: GCA_000005135.1) of Drosophila erecta. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

In a wide variety of animals, developmental crowding results in adults with smaller bodies. The crowding effect on body size in Drosophila melanogaster is canonically attributed to heightened competition for nutrition. However, whether other consequences of crowding also contribute to its effect on size remains an open question. We tested the relative contributions of nutritional competition, oxygen availability, and larval-generated metabolites to the crowding effect on size. We found that while nutrition explains most of the variation in body size due to crowding, oxygen also contributes in a sex- and nutrition-dependent manner. We found no evidence that larval-generated chemicals affect body size. These data confirm a widely suspected but untested role of nutrition in producing the crowding effect on size in D. melanogaster, while revealing an unexpected role of oxygen, and raise the possibility that behavior may be a mediator of density-dependent plasticity. Research HighlightsWe found that both nutrition and oxygen mediate the crowding effect on size in Drosophila melanogaster.

Gene model for the ortholog of tango (tgo) in the May 2011 (Broad dper_caf1/DperCAF1) Genome Assembly (GenBank Accession: GCA_000005195.1) of Drosophila persimilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Genomic selection can increase the rate of genetic gain in crop breeding programs, but its effectiveness depends on the reliability of phenotypic data, the size and composition of the training population (TP), and the statistical model used to estimate genomic breeding values. These design choices are especially important in resource-limited breeding programs, where additional replication, larger TPs, and more extensive genotyping compete for the same resources. Using empirical data from a sweet sorghum [Sorghum bicolor (L.) Moench] breeding population, developed by CHIBAS, we evaluated the effects of phenotyping replication, TP size, training-validation genomic relatedness, and genomic prediction (GP) model on predictive ability (PA). Grain yield, plant height, stem weight, and total soluble solids were evaluated across three field environments. Few studies in sorghum have examined these factors together with comparable empirical rigor. Increasing replication improved genomic heritability and PA for all traits and environments, with the largest gains observed for grain yield. Larger TPs and increased training-validation genomic relatedness also improved PA, but their effects were most significant when phenotype estimates were based on multiple replicates. GP models showed largely comparable PAs across all evaluated traits. Different models produced similar PA, with a few exceptions. These findings provide practical guidance for optimizing genomic selection in resource-limited sorghum breeding programs. ARTICLE SUMMARYGenomic selection can accelerate breeding only when the phenotypes used to train prediction models have high reliability. Using a sweet sorghum breeding population evaluated in three Haitian field environments, we quantified how replication number, training population size, training-validation genomic relatedness, and prediction model affected genomic predictive ability for grain yield, plant height, stem weight, and total soluble solids. Replication increased genomic heritability and predictive ability for all traits, with the strongest effects for grain yield. Larger and more connected training populations improved prediction, mainly when replication was adequate. These results provide practical guidance for resource-limited breeding programs. Core ideasO_LIIn this empirical sweet sorghum breeding population, phenotyping replication was the dominant factor explaining variation in genomic predictive ability across traits and environments. C_LIO_LIThe benefit of larger training populations and greater training-validation genomic relatedness increased when phenotype estimates were based on more replicates. C_LIO_LIGrain yield, the most environmentally sensitive trait evaluated, showed the largest response to improved replication and training-population design. C_LIO_LIBayesian models, rrBLUP, and GBLUP showed similar predictive abilities across traits and environments, suggesting that phenotyping and experimental design may be more important than model complexity. C_LI

During embryonic development bipotential gonads differentiate into either testes or ovaries under the direction of mutually exclusive gene networks. DNA methylation has been shown to regulate gene expression and to play a role in many developmental processes. This includes sex differentiation in which DNA methylation is linked to control of key sex genes. Whether DNA methylation regulates sexual differentiation in species where environmental influences such as temperature are involved is less clear. We conducted a genome-wide study in embryonic gonads of the central bearded dragon (Pogona vitticeps), a lizard with temperature induced sex reversal, and compared DNA methylation patterns to gene expression profiles at a stage of early sex differentiation. Overall, sex reversed ZZf females were found to have lower global methylation than both canonical sexes, ZZm males and ZWf females. We found that the expression of a key gene in sex differentiation, Amh, is regulated via DNA methylation. Amh is a driver of testis differentiation and is repressed in genetically determined females, as well as in temperature sex-reversed females, by hypermethylation around its transcription start site. Although the trigger of ovary determination is different in both groups of females, one by genetic complement and one by a temperature signal, downstream regulation of gene expression converges and seems to follow similar mechanisms.

Gene model for the ortholog of raptor in the D. eugracilis Apr. 2013 (BCM-HGSC/Deug_2.0) (DeugGB2) Genome Assembly (GenBank Accession: GCA_000236325.2) of Drosophila eugracilis. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

The rat tapeworm, Hymenolepis diminuta, is an important laboratory model for uncovering molecular processes that underly the success of tapeworms as parasites. Despite its importance, a high-quality reference genome for this species is lacking. Here we present a highly contiguous and effectively complete genome of H. diminuta assembled from PacBio HiFi long-read sequencing data. Our primary assembly consists of 7 scaffolds (N50=29.25 Mb) with total length of 186.53 Mb, has only 7 gaps, and contains 95.7% complete Lophotrochozoan BUSCOs. Our assembly allows us to confirm aspects of Hymenolepis genome organization, such as high repeat content and unusual chromosomal ends, and to show that Hymenolepis genomes encode [~]10,000 genes. Together with annotations of nuclear tRNAs, mtDNA protein coding genes, and mtDNA tRNAs, our assembly currently provides one of the most complete genome resources for a tapeworm species and will enable research on parasitism, animal regeneration, development, and evolution.