The regulatory network of the White Collar complex during early mushroom development in Schizophyllum commune.

Blue light is an important signal for fungal development. In the mushroom-forming basidiomycete Schizophyllum commune, blue light is detected by the White Collar complex, which consists of WC-1 and WC-2. Most of our knowledge on this complex is derived from the ascomycete Neurospora crassa, where both WC-1 and WC-2 contain GATA zinc-finger transcription factor domains. In basidiomycetes, WC-1 is truncated and does not contain a transcription factor domain, but both WC-1 and WC-2 are still important for development. We show that dimerization of WC-1 and WC-2 happens independent of light in S. commune, but that induction by light is required for promoter binding by the White Collar complex. Furthermore, the White Collar complex is a promoter of transcription, but binding of the complex alone is not always sufficient to initiate transcription. For its function, the White Collar complex associates directly with the promoters of structural genes involved in mushroom development, like hydrophobins, but also promotes the expression of other transcription factors that play a role in mushroom development.

Genome annotations for the ascomycete fungi Trichoderma harzianum, Trichoderma aggressivum, and Purpureocillium lilacinum.

We sequenced and annotated the genomes of the ascomycete fungi Trichoderma harzianum, Trichoderma aggressivum f. europaeum, and Purpureocillium lilacinum. Moreover, we developed a website to allow users to interactively analyze the assemblies, gene predictions, and functional annotations of these species and 70+ previously sequenced fungi.

Compatible solutes determine the heat resistance of conidia.

BACKGROUND: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. RESULTS: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. CONCLUSIONS: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales.

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

High sorbic acid resistance of Penicillium roqueforti is mediated by the SORBUS gene cluster.

Penicillium roqueforti is a major food-spoilage fungus known for its high resistance to the food preservative sorbic acid. Here, we demonstrate that the minimum inhibitory concentration of undissociated sorbic acid (MICu) ranges between 4.2 and 21.2 mM when 34 P. roqueforti strains were grown on malt extract broth. A genome-wide association study revealed that the six most resistant strains contained the 180 kbp gene cluster SORBUS, which was absent in the other 28 strains. In addition, a SNP analysis revealed five genes outside the SORBUS cluster that may be linked to sorbic acid resistance. A partial SORBUS knock-out (>100 of 180 kbp) in a resistant strain reduced sorbic acid resistance to similar levels as observed in the sensitive strains. Whole genome transcriptome analysis revealed a small set of genes present in both resistant and sensitive P. roqueforti strains that were differentially expressed in the presence of the weak acid. These genes could explain why P. roqueforti is more resistant to sorbic acid when compared to other fungi, even in the absence of the SORBUS cluster. Together, the MICu of 21.2 mM makes P. roqueforti among the most sorbic acid-resistant fungi, if not the most resistant fungus, which is mediated by the SORBUS gene cluster.

The Transcription Factor Roc1 Is a Key Regulator of Cellulose Degradation in the Wood-Decaying Mushroom Schizophyllum commune.

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δroc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune. Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

Genome sequences of 24 Aspergillus niger sensu stricto strains to study strain diversity, heterokaryon compatibility, and sexual reproduction.

Mating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24 strains were sequenced and analyzed revealing an average of 6.1 ± 2.0 variants/kb between Aspergillus niger sensu stricto strains. The genome sequences were used together with available genome data to generate a phylogenetic tree revealing 3 distinct clades within Aspergillus niger sensu stricto. The phylogenetic tree revealed that both MAT1-1 and MAT1-2 mating types were present in each of the 3 clades. The phylogenetic differences were used to select for strains to analyze heterokaryon compatibility. Conidial color markers (fwnA and brnA) and auxotrophic markers (pyrG and nicB) were introduced via CRISPR/Cas9-based genome editing in a selection of strains. Twenty-three parasexual crosses using 11 different strains were performed. Only a single parasexual cross between genetically highly similar strains resulted in a successful formation of heterokaryotic mycelium and subsequent diploid formation, indicating widespread heterokaryon incompatibility as well as multiple active heterokaryon incompatibility systems between Aspergillus niger sensu stricto strains. The 2 vegetatively compatible strains were of 2 different mating types and a stable diploid was isolated from this heterokaryon. Sclerotium formation was induced on agar media containing Triton X-100; however, the sclerotia remained sterile and no ascospores were observed. Nevertheless, this is the first report of a diploid Aspergillus niger sensu stricto strain with 2 different mating types, which offers the unique possibility to screen for conditions that might lead to ascospore formation in A. niger.

H3K4me2 ChIP-Seq reveals the epigenetic landscape during mushroom formation and novel developmental regulators of Schizophyllum commune.

Mushroom formation represents the most complex multicellular development in fungi. In the model mushroom Schizophyllum commune, comparative genomics and transcriptomics have previously resulted in a regulatory model of mushroom development. However, little is known about the role of epigenetic regulation. We used chromatin immunoprecipitation sequencing (ChIP-Seq) to determine the distribution of dimethylation of lysine 4 on histone H3 (H3K4me2), a mark for transcriptionally active genes, during monokaryotic and dikaryotic development. We identified a total of 6032 and 5889 sites during monokaryotic and dikaryotic development, respectively. The sites were strongly enriched near translation initiation sites of genes. Although the overall epigenetic landscape was similar between both conditions, we identified 837 sites of differential enrichment during monokaryotic or dikaryotic development, associated with 965 genes. Six transcription factor genes were enriched in H3K4me2 during dikaryotic development, indicating that these are epigenetically regulated during development. Deletion of two of these genes (fst1 and zfc7) resulted in arrested development of fruiting bodies, resulting in immature mushrooms. Together these results indicate that H3K4me2 ChIP-Seq is a powerful new tool to map the restructuring of the epigenetic landscape during mushroom development. Moreover, it can be used to identify novel developmental regulators.

Cycling in degradation of organic polymers and uptake of nutrients by a litter-degrading fungus.

Wood and litter degrading fungi are the main decomposers of lignocellulose and thus play a key role in carbon cycling in nature. Here, we provide evidence for a novel lignocellulose degradation strategy employed by the litter degrading fungus Agaricus bisporus (known as the white button mushroom). Fusion of hyphae allows this fungus to synchronize the activity of its mycelium over large distances (50 cm). The synchronized activity has a 13-h interval that increases to 20 h before becoming irregular and it is associated with a 3.5-fold increase in respiration, while compost temperature increases up to 2°C. Transcriptomic analysis of this burst-like phenomenon supports a cyclic degradation of lignin, deconstruction of (hemi-) cellulose and microbial cell wall polymers, and uptake of degradation products during vegetative growth of A. bisporus. Cycling in expression of the ligninolytic system, of enzymes involved in saccharification, and of proteins involved in nutrient uptake is proposed to provide an efficient way for degradation of substrates such as litter.

Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

Telomere-to-telomere assembled and centromere annotated genomes of the two main subspecies of the button mushroom Agaricus bisporus reveal especially polymorphic chromosome ends.

Agaricus bisporus, the most cultivated edible mushroom worldwide, is represented mainly by the subspecies var. bisporus and var. burnettii. var. bisporus has a secondarily homothallic life cycle with recombination restricted to chromosome ends, while var. burnettii is heterothallic with recombination seemingly equally distributed over the chromosomes. To better understand the relationship between genomic make-up and different lifestyles, we have de novo sequenced a burnettii homokaryon and synchronised gene annotations with updated versions of the published genomes of var. bisporus. The genomes were assembled into telomere-to-telomere chromosomes and a consistent set of gene predictions was generated. The genomes of both subspecies were largely co-linear, and especially the chromosome ends differed in gene model content between the two subspecies. A single large cluster of repeats was found on each chromosome at the same respective position in all strains, harbouring nearly 50% of all repeats and likely representing centromeres. Repeats were all heavily methylated. Finally, a mapping population of var. burnettii confirmed an even distribution of crossovers in meiosis, contrasting the recombination landscape of var. bisporus. The new findings using the exceptionally complete and well annotated genomes of this basidiomycete demonstrate the importance for unravelling genetic components underlying the different life cycles.

Impact of maturation and growth temperature on cell-size distribution, heat-resistance, compatible solute composition and transcription profiles of Penicillium roqueforti conidia.

Penicillium roqueforti is a major cause of fungal food spoilage. Its conidia are the main dispersal structures of this fungus and therefore the main cause of food contamination. These stress resistant asexual spores can be killed by preservation methods such as heat treatment. Here, the effects of cultivation time and temperature on thermal resistance of P. roqueforti conidia were studied. To this end, cultures were grown for 3, 5, 7 and 10 days at 25 °C or for 7 days at 15, 25 and 30 °C. Conidia of 3- and 10-day-old cultures that had been grown at 25 °C had D56-values of 1.99 ± 0.15 min and 5.31 ± 1.04 min, respectively. The effect of cultivation temperature was most pronounced between P. roqueforti conidia cultured for 7 days at 15 °C and 30 °C, where D56-values of 1.12 ± 0.05 min and 4.19 ± 0.11 min were found, respectively. Notably, D56-values were not higher when increasing both cultivation time and temperature by growing for 10 days at 30 °C. A correlation was found between heat resistance of conidia and levels of trehalose and arabitol, while this was not found for glycerol, mannitol and erythritol. RNA-sequencing showed that the expression profiles of conidia of 3- to 10-day-old cultures that had been grown at 25 °C were distinct from conidia that had been formed at 15 °C and 30 °C for 7 days. Only 33 genes were upregulated at both prolonged incubation time and increased growth temperature. Their encoded proteins as well as trehalose and arabitol may form the core of heat resistance of P. roqueforti conidia.

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype.

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context.

Genetic Underpinnings of Host Manipulation by Ophiocordyceps as Revealed by Comparative Transcriptomics.

Ant-infecting Ophiocordyceps fungi are globally distributed, host manipulating, specialist parasites that drive aberrant behaviors in infected ants, at a lethal cost to the host. An apparent increase in activity and wandering behaviors precedes a final summiting and biting behavior onto vegetation, which positions the manipulated ant in a site beneficial for fungal growth and transmission. We investigated the genetic underpinnings of host manipulation by: (i) producing a high-quality hybrid assembly and annotation of the Ophiocordyceps camponoti-floridani genome, (ii) conducting laboratory infections coupled with RNAseq of O. camponoti-floridani and its host, Camponotus floridanus, and (iii) comparing these data to RNAseq data of Ophiocordyceps kimflemingiae and Camponotus castaneus as a powerful method to identify gene expression patterns that suggest shared behavioral manipulation mechanisms across Ophiocordyceps-ant species interactions. We propose differentially expressed genes tied to ant neurobiology, odor response, circadian rhythms, and foraging behavior may result by activity of putative fungal effectors such as enterotoxins, aflatrem, and mechanisms disrupting feeding behaviors in the ant.

High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins.

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

Megaphylogeny resolves global patterns of mushroom evolution.

Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.

Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi.

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms.

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose.

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose.IMPORTANCEAspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

Genomics and Development of Lentinus tigrinus: A White-Rot Wood-Decaying Mushroom with Dimorphic Fruiting Bodies.

Lentinus tigrinus is a species of wood-decaying fungi (Polyporales) that has an agaricoid form (a gilled mushroom) and a secotioid form (puffball-like, with enclosed spore-bearing structures). Previous studies suggested that the secotioid form is conferred by a recessive allele of a single locus. We sequenced the genomes of one agaricoid (Aga) strain and one secotioid (Sec) strain (39.53-39.88 Mb, with 15,581-15,380 genes, respectively). We mated the Sec and Aga monokaryons, genotyped the progeny, and performed bulked segregant analysis (BSA). We also fruited three Sec/Sec and three Aga/Aga dikaryons, and sampled transcriptomes at four developmental stages. Using BSA, we identified 105 top candidate genes with nonsynonymous SNPs that cosegregate with fruiting body phenotype. Transcriptome analyses of Sec/Sec versus Aga/Aga dikaryons identified 907 differentially expressed genes (DEGs) along four developmental stages. On the basis of BSA and DEGs, the top 25 candidate genes related to fruiting body development span 1.5 Mb (4% of the genome), possibly on a single chromosome, although the precise locus that controls the secotioid phenotype is unresolved. The top candidates include genes encoding a cytochrome P450 and an ATP-dependent RNA helicase, which may play a role in development, based on studies in other fungi.