Dapeng Bao

Sequencing and comparative analysis of the straw mushroom (Volvariella volvacea) genome.

Volvariella volvacea, the edible straw mushroom, is a highly nutritious food source that is widely cultivated on a commercial scale in many parts of Asia using agricultural wastes (rice straw, cotton wastes) as growth substrates. However, developments in V. volvacea cultivation have been limited due to a low biological efficiency (i.e. conversion of growth substrate to mushroom fruit bodies), sensitivity to low temperatures, and an unclear sexuality pattern that has restricted the breeding of improved strains. We have now sequenced the genome of V. volvacea and assembled it into 62 scaffolds with a total genome size of 35.7 megabases (Mb), containing 11,084 predicted gene models. Comparative analyses were performed with the model species in basidiomycete on mating type system, carbohydrate active enzymes, and fungal oxidative lignin enzymes. We also studied transcriptional regulation of the response to low temperature (4°C). We found that the genome of V. volvacea has many genes that code for enzymes, which are involved in the degradation of cellulose, hemicellulose, and pectin. The molecular genetics of the mating type system in V. volvacea was also found to be similar to the bipolar system in basidiomycetes, suggesting that it is secondary homothallism. Sensitivity to low temperatures could be due to the lack of the initiation of the biosynthesis of unsaturated fatty acids, trehalose and glycogen biosyntheses in this mushroom. Genome sequencing of V. volvacea has improved our understanding of the biological characteristics related to the degradation of the cultivating compost consisting of agricultural waste, the sexual reproduction mechanism, and the sensitivity to low temperatures at the molecular level which in turn will enable us to increase the industrial production of this mushroom.

Microsatellites in the Genome of the Edible Mushroom, Volvariella volvacea

Using bioinformatics software and database, we have characterized the microsatellite pattern in the V. volvacea genome and compared it with microsatellite patterns found in the genomes of four other edible fungi: Coprinopsis cinerea, Schizophyllum commune, Agaricus bisporus, and Pleurotus ostreatus. A total of 1346 microsatellites have been identified, with mono-nucleotides being the most frequent motif. The relative abundance of microsatellites was lower in coding regions with 21 No./Mb. However, the microsatellites in the V. volvacea gene models showed a greater tendency to be located in the CDS regions. There was also a higher preponderance of trinucleotide repeats, especially in the kinase genes, which implied a possible role in phenotypic variation. Among the five fungal genomes, microsatellite abundance appeared to be unrelated to genome size. Furthermore, the short motifs (mono- to tri-nucleotides) outnumbered other categories although these differed in proportion. Data analysis indicated a possible relationship between the most frequent microsatellite types and the genetic distance between the five fungal genomes.

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating.

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.

The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay

Pleurotus eryngii (DC.) Quél. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.

A new approach for breeding low‐temperature‐resistant Volvariella volvacea strains: Genome shuffling in edible fungi

Volvariella volvacea is difficult to store fresh because of the lack of low‐temperature resistance. Many traditional mutagenic strategies have been applied in order to select out strains resistant to low temperature, but few commercially efficient strains have been produced. In order to break through the bottleneck of traditional breeding and significantly improve low‐temperature resistance of the edible fungus V. volvacea, strains resistant to low temperature were constructed by genome shuffling. The optimum conditions of V. volvacea strain mutation, protoplast regeneration, and fusion were determined. After protoplasts were treated with 1% (v/v) ethylmethylsulfonate (EMS), 40 Sec of ultraviolet (UV) irradiation, 600 Gy electron beam implantation, and 750 Gy60Co‐γ irradiation, separately, the lethality was within 70%–80%, which favored generating protoplasts being used in following forward mutation. Under these conditions, 16 strains of V. volvacea mutated by EMS, electron beam, UV irradiation, and 60Co‐γ irradiation were obtained. The 16 mutated protoplasts were selected to serve as the shuffling pool based on their excellent low‐temperature resistance. After four rounds of genome shuffling and low‐temperature resistance testing, three strains (VF1, VF2, and VF3) with high genetic stability were screened. VF1, VF2, and VF3 significantly enhanced fruit body shelf life to 20, 28, and 28 H at 10 °C, respectively, which exceeded 25%, 75%, and 75%, respectively, compared with the storage time of V23, the most low‐temperature‐resistant strain. Genome shuffling greatly improved the low‐temperature resistance of V. volvacea, and shortened the course of screening required to generate desirable strains. To our knowledge, this is the first paper to apply genome shuffling to breeding new varieties of mushroom, and offers a new approach for breeding edible fungi with optimized phenotype.

The complete mitochondrial genome of the widely cultivated edible fungus Lentinula edodes

Abstract The complete mitochondrial genome of the widely cultivated edible fungus Lentinula edodes was determined using the next-generation sequencing technology. The circular molecule is 116,819 bp in length with a GC content of 30.75%. Conserved genes including 13 putative protein-coding genes and 24 tRNAs were located on the same strand. We detected 14 introns invading 4 genes, including cob, cox1, nad1, and nad5. The phylogenetic analysis confirmed that L. edodes was a number of Agaricales. This mitochondrial genome may open new avenues for understanding the phylogeny and evolution of Omphalotaceae and Agaricales.

A newly discovered ubiquitin-conjugating enzyme E2 correlated with the cryogenic autolysis of Volvariella volvacea.

In Volvariella volvacea, a species of edible mushroom, cryogenic autolysis is a typical part of abnormal metabolism. Previous functional annotation cluster analyses of cold-induced gene expression profiles have shown that the ubiquitin-conjugating enzyme E2 (UBE2), rather than the cyclin-like F-box domain alone, forms the functional cluster. In this study, analysis of gene expression profiling showed that only one type of UBE2 in V. volvacea (UBEV2) was significantly up-regulated. Further quantitative real-time PCR analysis confirmed that the expression of UBEV2 was significantly up-regulated (P<0.05) after cold-treatment lasting 4, 6, and 8h. This provided evidence that UBEV2 was closely correlated with cryogenic autolysis. The specific distribution of UBEV2 in recently diverged herb decay fungi indicated that UBEV2 was not evolutionarily correlated with early diverging fungi. Phylogenetic analysis indicated that UBEV2 was generated by horizontal gene transfer (HGT) from the ancestry of Selaginella moellendorffii UBE2. Further relative time estimation and detection of natural selection showed that there has been recent positive selection after HGT in UBEV2. Molecular modeling and logo analysis showed that the cysteine-cysteine motif is the characteristic of the UBEV2 family. These observations indicate that UBEV2 is a new type of UBE2 correlated with the cryogenic autolysis of V. volvacea.

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing

The nuclear ribosomal DNA internal transcribed spacer (ITS) has been widely used to assess the fungal composition in different environments by deep sequencing. To evaluate the ITS in the analysis of fungal diversity, comparisons of the clustering and taxonomy generated by sequencing with different portions of the whole fragment were conducted in this study. For a total of 83,120 full-length ITS sequences obtained from the UNITE database, it was found that, on average, ITS1 varied more than ITS2 within the kingdom Fungi; this variation included length and GC content variations and polymorphisms, with some polymorphisms specific to particular fungal groups. The taxonomic accuracy for ITS was higher than that for ITS1 or ITS2. The commonly used operational taxonomic unit (OTU) for evaluating fungal diversity and richness assigned several species to a single OTU even with clustering at 99.00% sequence similarity. The clustering and taxonomic capacities did not differ between ITS1 and ITS2. However, the OTU commonality between ITS1 and ITS2 was very low. To test this observation further, 219,741 pyrosequencing reads, including 39,840 full-length ITS sequences, were obtained from 10 soil samples and were clustered into OTUs. The pyrosequencing results agreed with the results of the in silico analysis. ITS1 might overestimate the fungal diversity and richness. Analyses using ITS, ITS1 and ITS2 yielded several different taxa, and the taxonomic preferences for ITS and ITS2 were similar. The results demonstrated that ITS2 alone might be a more suitable marker for revealing the operational taxonomic richness and taxonomy specifics of fungal communities when the full-length ITS is not available.

Three complete mitochondrial genomes of straw-rotting edible fungus Volvariella volvacea using next generation sequencing

Abstract The straw-rotting edible fungus Volvariella volvacea is a widely cultivated edible fungus across China and Southeast Asian countries. Three complete mitochondrial genomes of V. volvacea from China, Thailand, and India were determined using the next-generation sequencing technology. The genome sizes of the three strains (China, Thailand, and India) were 62,541 bp, 64,531 bp, and 65,668 bp with GC contents of 38.46%, 38.56%, and 38.52%, respectively. All the genomes encoded 14 conserved protein-coding genes, the small ribosomal RNA subunits (rns), large ribosomal RNA subunits (rnl), and 23 tRNAs were located on the same strand. In the putative protein-coding genes, four introns were distributed in cox1 in the genomes of V23-1 and V8. 5 introns (four introns invaded into cox1and one intron invaded into cob) were detected in Tai8. The phylogenetic analysis confirmed that V. volvacea was a number of Agaricales. This mitochondrial genome may open new avenues for understanding the phylogeny and evolution of Pluteaceae and Agaricales.