No abstract

Genetic marker technology designed to detect naturally occurring polymorphisms at the DNA level had become an invaluable and revolutionizing tool for both applied and basic studies of fungi. To eliminate the confusion on the taxonomy of Ganoderma strains, in this study, a collection of 31 accessions representative of morphotypes and some unclassified types was used for analyzing molecular diversity using a novel molecular marker sequence-related amplified polymorphism (SRAP). This collection included commercial cultivars and wild varieties that represented the great diversification of types from different countries and regions. The experimental results showed that 50 out of 95 combinations of primers turned out to be polymorphic, and 85 polymorphism bands were obtained using six combinations. Based on the appearances of markers, the genetic similarity coefficients were calculated, and genetic variations were observed (0 approximately 1) among the 31 different Ganoderma strains. The group of Ganoderma lucidum showed significant differences from the group of Ganoderma sinense. Moreover, G. lucidum in China was also different from G. lucidum in Yugoslavia. At the same time, cluster analysis successfully categorized these 31 Ganoderma strains into five groups. These results revealed the genetic diversity of Ganoderma strains and their correlation with geographic environments. It also suggested SRAP marker could be used in the taxonomic analysis of fungi. To our knowledge, this is the first application of SRAP marker on the systematics of Ganoderma strains within basidiomycetes.

The initiation of sexual development in the important edible and medicinal mushroom Flammulina velutipes is controlled by special genes at two different, independent, mating type (MAT) loci: HD and PR. We expanded our understanding of the F. velutipes mating type system by analyzing the MAT loci from a series of strains. The HD locus of F. velutipes houses homeodomain genes (Hd genes) on two separated locations: sublocus HD-a and HD-b. The HD-b subloci contained strain-specific Hd1/Hd2 gene pairs, and crosses between strains with different HD-b subloci indicated a role in mating. The function of the HD-a sublocus remained undecided. Many, but not all strains contained the same conserved Hd2 gene at the HD-a sublocus. The HD locus usually segregated as a whole, though we did detect one new HD locus with a HD-a sublocus from one parental strain, and a HD-b sublocus from the other. The PR locus of F. velutipes contained pheromone receptor (STE3) and pheromone precursor (Pp) genes at two locations, sublocus PR-a and PR-b. PR-a and PR-b both contained sets of strain-specific STE3 and Pp genes, indicating a role in mating. PR-a and PR-b cosegregated in our experiments. However, the identification of additional strains with identical PR-a, yet different PR-b subloci, demonstrated that PR subloci can recombine within the PR locus. In conclusion, at least three of the four MAT subloci seem to participate in mating, and new HD and PR loci can be generated through intralocus recombination in F. velutipes.

c Antifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress, and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of the transcription factor gene ccg-8, which is known to be a clock-controlled gene, made

Antifungal azoles inhibit ergosterol biosynthesis by interfering with lanosterol 14α-demethylase. In this study, seven upregulated and four downregulated ergosterol biosynthesis genes in response to ketoconazole treatment were identified in Neurospora crassa. Azole sensitivity test of knockout mutants for six ketoconazole-upregulated genes in ergosterol biosynthesis revealed that deletion of only sterol C-22 desaturase ERG5 altered sensitivity to azoles: the erg5 mutant was hypersensitive to azoles but had no obvious defects in growth and development. The erg5 mutant accumulated higher levels of ergosta 5,7-dienol relative to the wild type but its levels of 14α-methylated sterols were similar to the wild type. ERG5 homologs are highly conserved in fungal kingdom. Deletion of Fusarium verticillioides erg5 also increased ketoconazole sensitivity, suggesting that the roles of ERG5 homologs in azole resistance are highly conserved among different fungal species, and inhibition of ERG5 could reduce the usage of azoles and thus provide a new target for drug design.

The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.

Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium.

Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3′-tag digital gene expression (DGE) reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.