Chromosome-level assembly of Dictyophora rubrovolvata genome using third-generation DNA sequencing and Hi-C analysis.

Dictyophora rubrovolvata, a rare edible mushroom with both nutritional and medicinal values, was regarded as the "queen of the mushroom" for its attractive appearance. Dictyophora rubrovolvata has been widely cultivated in China in recent years, and many researchers were focusing on its nutrition, culture condition, and artificial cultivation. Due to a lack of genomic information, research on bioactive substances, cross breeding, lignocellulose degradation, and molecular biology is limited. In this study, we report a chromosome-level reference genome of D. rubrovolvata using the PacBio single-molecule real-time-sequencing technique and high-throughput chromosome conformation capture (Hi-C) technologies. A total of 1.83 Gb circular consensus sequencing reads representing ∼983.34 coverage of the D. rubrovolvata genome were generated. The final genome was assembled into 136 contigs with a total length of 32.89 Mb. The scaffold and contig N50 length were 2.71 and 2.48 Mb, respectively. After chromosome-level scaffolding, 11 chromosomes with a total length of 28.24 Mb were constructed. Genome annotation further revealed that 9.86% of the genome was composed of repetitive sequences, and a total of 508 noncoding RNA (rRNA: 329, tRNA: 150, ncRNA: 29) were annotated. In addition, 9,725 protein-coding genes were predicted, among which 8,830 (90.79%) genes were predicted using homology or RNA-seq. Benchmarking Universal Single-Copy Orthologs results further revealed that there were 80.34% complete single-copy fungal orthologs. In this study, a total of 360 genes were annotated as belonging to the carbohydrate-active enzymes family. Further analysis also predicted 425 cytochromes P450 genes, which can be classified into 41 families. This highly accurate, chromosome-level reference genome of D. rubrovolvata will provide essential genomic information for understanding the molecular mechanism in its fruiting body formation during morphological development and facilitate the exploitation of medicinal compounds produced by this mushroom.

Transcriptomic and Metabolomic Profiles Provide Insights into the Red-Stipe Symptom of Morel Fruiting Bodies.

The cultivation of true morels (Morchella spp., Morchellaceae, Ascomycota) has rapidly expanded in recent years, especially in China. Red stipe is a symptom wherein the stipe of morel fruiting bodies becomes red-gray, resulting in the gradual death of the affected fruiting bodies. The impact of red-stipe symptom occurrence on the development and nutritional quality of morel fruiting bodies remains unclear. Herein, morel ascocarps with the red-stipe symptom (R) and normal (N), artificially cultivated in the Fujian Province of China, were selected for the transcriptome and metabolome analysis to study the physiological and biochemical responses of morel fruiting bodies to the red-stipe symptom. Transcriptome data revealed several differentially expressed genes between the R and N groups significantly enriched in the tyrosine, riboflavin, and glycerophospholipid metabolism pathways. Similarly, the differentially accumulated metabolites were mainly assigned to metabolic pathways, including tyrosine, the biosynthesis of plant secondary metabolites, and the biosynthesis of amino acids. Moreover, the transcriptome and metabolome data combination revealed that tyrosine metabolism was the most enriched pathway, which was followed by ATP-binding cassette (ABC) transport, alanine, aspartate, and glutamate metabolism. Overall, the integration of transcriptomic and metabolomic data of M. sextelata affected by red-stipe symptoms identified several important genes, metabolites, and pathways. These findings further improve our understanding of the mechanisms underlying the red-stipe symptom development of M. sextelata and provide new insights into how to optimize its cultivation methods.

Comparative transcriptomics reveals unique pine wood decay strategies in the Sparassis latifolia.

Sparassis latifolia is a valuable edible mushroom, growing on fresh pine wood sawdust substrate. However, the mechanistic bases are poorly understood. The gene expression profiles of S. latifolia were analyzed from submerged cultures with fresh pine wood sawdust substrate for different time (0 h, 1 h, 6 h, 1 day, 5 days, and 10 days, respectively). The total number of differentially expressed genes (DEGs) identified under pine sawdust inducing was 2,659 compared to 0 h (CK). And 1,073, 520, 385, 424, and 257 DEGs were identified at the five time points, respectively. There were 34 genes in common at all inoculated time points, including FAD/NAD(P)-binding domain-containing protein, glucose methanol choline (GMC) oxidoreductase, flavin-containing monooxygenase, and taurine catabolism dioxygenase. Weighted gene co-expression analysis (WGCNA) was then used to compare the molecular characteristics among the groups and identified that the blue module had the highest correlation with the time induced by pine wood sawdust. There were 102 DEGs out of 125 genes in the blue model, which were most enriched in nitronate monooxygenase activity, dioxygenase activity, and oxidation-reduction process GO terms (p < 0.05), and peroxisome in KEGG pathway. This may provide clues into mechanisms that S. latifolia can grow on fresh pine wood sawdust substrate.

Chromosome-scale assembly of the Sparassis latifolia genome obtained using long-read and Hi-C sequencing.

Sparassis latifolia is a valuable edible mushroom cultivated in China. In 2018, our research group reported an incomplete and low-quality genome of S. latifolia obtained by Illumina HiSeq 2500 sequencing. These limitations in the available genome have constrained genetic and genomic studies in this mushroom resource. Herein, an updated draft genome sequence of S. latifolia was generated by Oxford Nanopore sequencing and the high-through chromosome conformation capture (Hi-C) technique. A total of 8.24 Gb of Oxford Nanopore long reads representing ∼198.08X coverage of the S. latifolia genome were generated. Subsequently, a high-quality genome of 41.41 Mb, with scaffold and contig N50 sizes of 3.31 and 1.51 Mb, respectively, was assembled. Hi-C scaffolding of the genome resulted in 12 pseudochromosomes containing 93.56% of the bases in the assembled genome. Genome annotation further revealed that 17.47% of the genome was composed of repetitive sequences. In addition, 13,103 protein-coding genes were predicted, among which 98.72% were functionally annotated. BUSCO assay results further revealed that there were 92.07% complete BUSCOs. The improved chromosome-scale assembly and genome features described here will aid further molecular elucidation of various traits, breeding of S. latifolia, and evolutionary studies with related taxa.

Identification and Evaluation of Reference Genes for qRT-PCR Normalization in Sparassis latifolia (Agaricomycetes).

Real-time quantitative polymerase chain reaction (qRT-PCR) has emerged as a powerful and popular tool for quantitating differences in transcriptional gene expression levels between samples. Validation of the stability of reference genes is a fundamental step before initiating qRT-PCR assays. Sparassis latifolia is an edible and medicinal fungus containing a remarkably high concentration of ß-glucan, which has many biological and pharmacologic activities. S. latifolia may be a model species for studying fungal photobiology because its fruiting body formation requires more light than other fungi. However, suitable reference genes for qRT-PCR have not yet been determined. In the present study, 10 candidate reference genes in S. latifolia were evaluated and validated under different developmental stages and light conditions. To evaluate the suitability of candidate reference genes, three popular software programs (geNorm, NormFinder, and BestKeeper), along with the delta Ct method, were used to analyze these genes; the final ranking was determined using RefFinder. According to our results, Actin and GAPDH were expressed at the most stable levels under different developmental stages and light conditions.

Integration of ATAC-Seq and RNA-Seq Identifies Key Genes in Light-Induced Primordia Formation of Sparassis latifolia.

Light is an essential environmental factor for Sparassis latifolia primordia formation, but the molecular mechanism is still unclear. In this study, differential expression profiling of light-induced primordia formation (LIPF) was established by integrating the assay for transposase accessible chromatin by sequencing (ATAC-seq) and RNA-seq technology. The integrated results from the ATAC-seq and RNA-seq showed 13 down-regulated genes and 17 up-regulated genes in both the L vs. D and P vs. D groups, for both methods. According to the gene ontology (GO) annotation of these differentially expressed genes (DEGs), the top three biological process categories were cysteine biosynthetic process via cystathionine, vitamin B6 catabolic, and glycine metabolic; the top three molecular function categories were 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase activity, glycine binding, and pyridoxal phosphate binding; cellular component categories were significantly enriched in the glycine cleavage complex. The KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis revealed that these genes were associated with vitamin B6 metabolism; selenocompound metabolism; cysteine and methionine metabolism; glycine, serine, and threonine metabolism; and glyoxylate and dicarboxylate metabolism pathways. The expression of most of the DEGs was validated by qRT-PCR. To the best of our knowledge, this study is the first integrative analysis of ATAC-seq and RNA-seq for macro-fungi. These results provided a new perspective on the understanding of key pathways and hub genes in LIPF in S. latifolia. It will be helpful in understanding the primary environmental response, and provides new information to the existing models of primordia formation in edible and medicinal fungi.

De Novo Sequencing of a Sparassis latifolia Genome and Its Associated Comparative Analyses.

Known to be rich in ß-glucan, Sparassis latifolia (S. latifolia) is a valuable edible fungus cultivated in East Asia. A few studies have suggested that S. latifolia is effective on antidiabetic, antihypertension, antitumor, and antiallergen medications. However, it is still unclear genetically why the fungus has these medical effects, which has become a key bottleneck for its further applications. To provide a better understanding of this fungus, we sequenced its whole genome, which has a total size of 48.13 megabases (Mb) and contains 12,471 predicted gene models. We then performed comparative and phylogenetic analyses, which indicate that S. latifolia is closely related to a few species in the antrodia clade including Fomitopsis pinicola, Wolfiporia cocos, Postia placenta, and Antrodia sinuosa. Finally, we annotated the predicted genes. Interestingly, the S. latifolia genome encodes most enzymes involved in carbohydrate and glycoconjugate metabolism and is also enriched in genes encoding enzymes critical to secondary metabolite biosynthesis and involved in indole, terpene, and type I polyketide pathways. As a conclusion, the genome content of S. latifolia sheds light on its genetic basis of the reported medicinal properties and could also be used as a reference genome for comparative studies on fungi.

p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor.

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.