Integrative analysis of genome and transcriptome reveal the genetic basis of high temperature tolerance in pleurotus giganteus (Berk. Karun & Hyde).

BACKGROUND: Pleurotus giganteus is a commonly cultivated mushroom with notable high temperature resistance, making it significant for the growth of the edible fungi industry in the tropics. Despite its practical importance,, the genetic mechanisms underlying its ability to withstand high temperature tolerance remain elusive. RESULTS: In this study, we performed high-quality genome sequencing of a monokaryon isolated from a thermotolerant strain of P. giganteus. The genome size was found to be 40.11 Mb, comprising 17 contigs and 13,054 protein-coding genes. Notably, some genes related to abiotic stress were identified in genome, such as genes regulating heat shock protein, protein kinase activity and signal transduction. These findings provide valuable insights into the genetic basis of P. giganteus' high temperature resistance. Furthermore, the phylogenetic tree showed that P. giganteus was more closely related to P. citrinopileatus than other Pleurotus species. The divergence time between Pleurotus and Lentinus was estimated as 153.9 Mya, and they have a divergence time with Panus at 168.3 Mya, which proved the taxonomic status of P. giganteus at the genome level. Additionally, a comparative transcriptome analysis was conducted between mycelia treated with 40 °C heat shock for 18 h (HS) and an untreated control group (CK). Among the 2,614 differentially expressed genes (DEGs), 1,303 genes were up-regulated and 1,311 were down-regulated in the HS group. The enrichment analysis showed that several genes related to abiotic stress, including heat shock protein, DnaJ protein homologue, ubiquitin protease, transcription factors, DNA mismatch repair proteins, and zinc finger proteins, were significantly up-regulated in the HS group. These genes may play important roles in the high temperature adaptation of P. giganteus. Six DEGs were selected according to fourfold expression changes and were validated by qRT-PCR, laying a good foundation for further gene function analysis. CONCLUSION: Our study successfully reported a high-quality genome of P. giganteus and identified genes associated with high-temperature tolerance through an integrative analysis of the genome and transcriptome. This study lays a crucial foundation for understanding the high-temperature tolerance mechanism of P. giganteus, providing valuable insights for genetic modification of P. giganteus strains and the development of high-temperature strains for the edible fungus industry, particularly in tropical regions.

Massive genome investigations reveal insights of prevalent introgression for environmental adaptation and triterpene biosynthesis in Ganoderma.

Genome introgression is one of the driving forces that can increase species and genetic diversity and facilitate the adaptive evolution of organisms and biodiversity conservation. However, the genomic introgression and its contribution to biodiversity of macrofungi are still unclear. The genus Ganoderma is a typical macrofungal group that plays crucial roles in forest ecosystem as saprophytic organisms and plant pathogens, and is also involved in human health as medicinal mushrooms. Most public Ganoderma genomes are fragmented, and reference genomes and whole-genome information of diverse germplasm resources for many Ganoderma species are lacking, thus hindering functional and evolutionary genomic investigations among Ganoderma species. In this study, we provide high-quality genomes of 10 Ganoderma species and whole-genome variants data of 224 individuals from various ecoregions, enabling us to infer the phylogeny of Ganoderma species and their historical population dynamics. Based on whole-genome variants, widespread and genome-wide introgression among Ganoderma species is revealed. Genes with significant introgression signals were related to stress response, digestive absorption, and secondary metabolite synthesis, factors that may contribute to environmental adaptation and important biocomponent metabolism. CYP512U6, an essential functional gene in the CYP450 family related to Ganoderma triterpene synthesis, was detected with significant introgression and selection signals combined with Ganoderma metabolomic analysis, indicating that both ancient gene exchange and recent domestication have contributed to the categories and content of secondary metabolites of Ganoderma. The reference genomes, whole-genome variants, and metabolite profiles could serve as abundant and valuable genetic resources for evolution, ecology, and conservation investigations of Ganoderma species and other macrofungi.

Diversity of Ascomycota in Jilin: Introducing Novel Woody Litter Taxa in Cucurbitariaceae.

Cucurbitariaceae has a high biodiversity worldwide on various hosts and is distributed in tropical and temperate regions. Woody litters collected in Changchun, Jilin Province, China, revealed a distinct collection of fungi in the family Cucurbitariaceae based on morphological and molecular data. Phylogenetic analyses of the concatenated matrix of the internal transcribed spacer (ITS) region, the large subunit (LSU) of ribosomal DNA, the RNA polymerase II subunit (rpb2), the translation elongation factor 1-alpha (tef1-α) and ß-tubulin (ß-tub) genes indicated that the isolates represent Allocucurbitaria and Parafenestella species based on maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis (BPP). We report four novel species: Allocucurbitaria mori, Parafenestella changchunensis, P. ulmi and P. ulmicola. The importance of five DNA markers for species-level identification in Cucurbitariaceae was determined by Assemble Species by Automatic Partitioning (ASAP) analyses. The protein-coding gene ß-tub is determined to be the best marker for species level identification in Cucurbitariaceae.

Pan-Genomes Provide Insights into the Genetic Basis of Auricularia heimuer Domestication.

In order to reveal the genetic variation signals of Auricularia heimuer that have occurred during their domestication and to find potential functional gene families, we constructed a monokaryotic pan-genome of A. heimuer representing four cultivated strains and four wild strains. The pan-genome contained 14,089 gene families, of which 67.56% were core gene families and 31.88% were dispensable gene families. We screened substrate utilization-related genes such as the chitinase gene ahchi1 of the glycoside hydrolase (GH) 18 family and a carbohydrate-binding module (CBM)-related gene from the dispensable families of cultivated populations. The genomic difference in the ahchi1 gene between the wild and cultivated genomes was caused by a 33 kb presence/absence variation (PAV). The detection rate of the ahchi1 gene was 93.75% in the cultivated population, significantly higher than that in the wild population (17.39%), indicating that it has been selected in cultivated strains. Principal component analysis (PCA) of the polymorphic markers in fragments near the ahchi1 gene was enriched in cultivated strains, and this was caused by multiple independent instances of artificial selection. We revealed for the first time the genetic basis of the ahchi1 gene in domestication, thereby providing a foundation for elucidating the potential function of the ahchi1 gene in the breeding of A. heimuer.

Genomic Features of Cladobotryum dendroides, Which Causes Cobweb Disease in Edible Mushrooms, and Identification of Genes Related to Pathogenicity and Mycoparasitism.

Cladobotryum dendroides, which causes cobweb disease in edible mushrooms, is one of the major fungal pathogens. Our previous studies focused on the genetic and morphological characterization of this fungus, as well as its pathogenicity and the identification of appropriate fungicides. However, little is known about the genome characters, pathogenic genes, and molecular pathogenic mechanisms of C. dendroides. Herein, we reported a high-quality de novo genomic sequence of C. dendroides and compared it with closely-related fungi. The assembled C. dendroides genome was 36.69 Mb, consisting of eight contigs, with an N50 of 4.76 Mb. This genome was similar in size to that of C. protrusum, and shared highly conserved syntenic blocks and a few inversions with C. protrusum. Phylogenetic analysis revealed that, within the Hypocreaceae, Cladobotryum was closer to Mycogone than to Trichoderma, which is consistent with phenotypic evidence. A significant number of the predicted expanded gene families were strongly associated with pathogenicity, virulence, and adaptation. Our findings will be instrumental for the understanding of fungi-fungi interactions, and for exploring efficient management strategies to control cobweb disease.

Genetic and Pathogenic Variability of Mycogone perniciosa Isolates Causing Wet Bubble Disease on Agaricus bisporus in China.

Wet bubble disease, caused by Mycogone perniciosa, is a major threat to Agaricus bisporus production in China. In order to understand the variability of in genetic, pathogenicity, morphology, and symptom production of the fungus, 18 isolates of the pathogen were collected from diseased A. bisporus in different provinces in China. The isolates were characterized by a combination of morphological, cultural, and molecular pathogenicity testing on different strains of A. bisporus and amplified fragment length polymorphism (AFLP) analysis. The 18 isolates were identified by Koch's postulate and confirmed different pathogenic variability among them. The yellow to brown isolates were more virulent than the white isolates. AFLP markers clustered the isolates into two distinct groups based on their colony color, with a high level of polymorphism of Jaccard similarities ranges from 0.39% to 0.64%. However, there was no evidence of an association between the genetic diversity and the geographical origin of the isolates. Through knowledge of the genetic diversity, phenotypic virulence of M. perniciosa is a key factor for successful breeding of resistant strains of A. bisporus and developing of an integrated disease management strategy to manage wet bubble disease of A. bisporus.

Genomic Analyses Provide Insights Into the Evolutionary History and Genetic Diversity of Auricularia Species.

Species in the genus Auricularia play important roles for people's food and nutrition especially Auricularia cornea and A. heimuer. To understand their evolutionary history, genome structure, and population-level genetic variation, we performed a high-quality genome sequencing of Auricularia cornea and the corresponding comparative genomic analysis. The genome size of A. cornea was similar to Auricularia subglabra, but 1.5 times larger than that of A. heimuer. Several factors were responsible for genome size variation including gene numbers, repetitive elements, and gene lengths. Phylogenomic analysis revealed that the estimated divergence time between A. heimuer and other Auricularia is ∼79.1 million years ago (Mya), while the divergence between A. cornea and A. subglabra occurred in ∼54.8 Mya. Population genomic analysis also provided insight into the demographic history of A. cornea and A. heimuer, indicating that their populations fluctuated over time with global climate change during Marine Isotope Stage 5-2. Moreover, despite the highly similar external morphologies of A. cornea and A. heimuer, their genomic properties were remarkably different. The A. cornea genome only shared 14% homologous syntenic blocks with A. heimuer and possessed more genes encoding carbohydrate-active enzymes and secondary metabolite biosynthesis proteins. The cross-taxa transferability rates of simple sequence repeat (SSR) and insertion or deletion (InDel) markers within the genus Auricularia were also lower than that previously observed for species within the same genus. Taken together, these results indicate a high level of genetic differentiation between these two Auricularia species. Consequently, our study provides new insights into the genomic evolution and genetic differentiation of Auricularia species that will facilitate future genetic breeding.

Pleurotus eryngii Genomes Reveal Evolution and Adaptation to the Gobi Desert Environment.

Pleurotus eryngii (King Oyster) is one of the most highly prized edible mushrooms. Among the diverse varieties within P. eryngii, P. eryngii var. eryngii is the commonest one, with a worldwide distribution, while P. eryngii var. ferulae is only distributed in Europe and China, and is especially adapted to the Gobi Desert in Xinjiang Autonomous Region of China. However, little is known about the genome-wide pattern of evolution and adaptation to the divergent environments of P. eryngii. Here, we present the high-quality genome sequences of P. eryngii var. eryngii strain PEE81 originating from Europe and P. eryngii var. ferulae strain PEF12 originating from the Gobi Desert of China. The assembled genome sizes of PEE81 and PEF12 were 53.6 and 48.0 Mbp, respectively, which are larger than other reported genomes in the genus Pleurotus. We propose that the selective amplification of long terminal repeat (LTR) retrotransposons increases the genome size of the genus Pleurotus, and may play a key role in driving their rapid species diversification. Molecular clock analyses of five Pleurotus species, namely PEE81, PEF12, P. tuoliensis, P. ostreatus and P. cf. floridanus suggest that the divergence estimates of the genus Pleurotus over time scales ranged from ∼4 to ∼38 million years ago (Mya), and PEE81 and PEF12 diverged at ∼13 Mya. The whole genome resequencing of 33 geographically diverse strains of P. eryngii var. eryngii and var. ferulae was then performed and the genome variation among and within these two populations were investigated. Comparative analyses of these two populations detected several candidate genes related to stress responses and DNA repair that are putatively involved in adaptation to the Gobi Desert environment. These findings offer insights into genome evolution of the genus Pleurotus and provide valuable genomic resources for King Oyster mushroom breeding.

Development of Novel Polymorphic EST-SSR Markers in Bailinggu (Pleurotus tuoliensis) for Crossbreeding.

Identification of monokaryons and their mating types and discrimination of hybrid offspring are key steps for the crossbreeding of Pleurotus tuoliensis (Bailinggu). However, conventional crossbreeding methods are troublesome and time consuming. Using RNA-seq technology, we developed new expressed sequence tag-simple sequence repeat (EST-SSR) markers for Bailinggu to easily and rapidly identify monokaryons and their mating types, genetic diversity and hybrid offspring. We identified 1110 potential EST-based SSR loci from a newly-sequenced Bailinggu transcriptome and then randomly selected 100 EST-SSRs for further validation. Results showed that 39, 43 and 34 novel EST-SSR markers successfully identified monokaryons from their parent dikaryons, differentiated two different mating types and discriminated F1 and F2 hybrid offspring, respectively. Furthermore, a total of 86 alleles were detected in 37 monokaryons using 18 highly informative EST-SSRs. The observed number of alleles per locus ranged from three to seven. Cluster analysis revealed that these monokaryons have a relatively high level of genetic diversity. Transfer rates of the EST-SSRs in the monokaryons of closely-related species Pleurotuseryngii var. ferulae and Pleurotus ostreatus were 72% and 64%, respectively. Therefore, our study provides new SSR markers and an efficient method to enhance the crossbreeding of Bailinggu and closely-related species.

Comparative Transcriptome Analysis Identified Candidate Genes Related to Bailinggu Mushroom Formation and Genetic Markers for Genetic Analyses and Breeding.

Bailinggu (Pleurotus tuoliensis) is a major, commercially cultivated mushroom and widely used for nutritional, medicinal, and industrial applications. Yet, the mushroom's genetic architecture and the molecular mechanisms underlying its formation are largely unknown. Here we performed comparative transcriptomic analysis during Bailinggu's mycelia, primordia, and fruiting body stages to identify genes regulating fruiting body development and develop EST-SSR markers assessing the genetic value of breeding materials. The stage-specific and differentially expressed unigenes (DEGs) involved in morphogenesis, primary carbohydrate metabolism, cold stimulation and blue-light response were identified using GO and KEGG databases. These unigenes might help Bailinggu adapt to genetic and environmental factors that influence fructification. The most pronounced change in gene expression occurred during the vegetative-to-reproductive transition, suggesting that is most active and key for Bailinggu development. We then developed 26 polymorphic and informative EST-SSR markers to assess the genetic diversity in 82 strains of Bailinggu breeding materials. These EST-SSRs exhibited high transferability in closely related species P. eryngii var. ferulae and var. eryngii. Genetic population structure analysis indicated that China's Bailinggu has low introgression with these two varieties and likely evolved independently. These findings provide new genes, SSR markers, and germplasm to enhance the breeding of commercially cultivated Bailinggu.

Jianpi Qingchang decoction alleviates ulcerative colitis by inhibiting nuclear factor-κB activation.

AIM: To investigate the therapeutic effect of Jianpi Qingchang decoction (JPQCD) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. METHODS: C57BL/c mice were injected intragastrically with 5% DSS instead of drinking water for 7 d, and their body weight, diarrhea severity and fecal bleeding were monitored, while the mice in the control group were treated with standard drinking water, without DSS. After 7 d, the DSS drinking water was changed to normal water and the DSS group continued with DSS water. The control and DSS groups were given normal saline by intragastric injection. The 5-aminosalicylic acid (5-ASA) group was treated orally with 5-ASA at a dose of 100 mg/kg daily. The JPQCD group was treated orally with JPQCD at a dose of 17.1 g/kg daily. On day 14, the colon length was measured, the colorectal histopathological damage score was assessed, and protein levels of interleukin (IL)-1ß, IL-8 and tumor necrosis factor-alpha (TNF-α) in colon supernatants were measured by enzyme-linked immunosorbent assay. mRNA expression of IL-1ß, IL-8, TNF-α and nuclear factor-kappa B (NF-κB) was detected by real-time quantitative polymerase chain reaction. Western blotting was used to detect the protein expression of NF-κB and inhibitor of kappa B. RESULTS: Acute inflammation occurred in the mice administered DSS, including the symptoms of losing body weight, loose feces/watery diarrhea and presence of fecal blood; all these symptoms worsened at 7 d. The colons of mice treated with DSS were assessed by histological examination, and the results confirmed that acute inflammation had occurred, as evidenced by loss of colonic mucosa and chronic inflammatory cell infiltration, and these features extended into the deeper layer of the colon walls. The expression levels of IL-1ß, IL-8 and TNF-α in the DSS group were higher than those in the control group (P < 0.05), and the expression levels of IL-1ß, IL-8 and TNF-α in the JPQCD and 5-ASA groups were lower than those in the DSS group after treating with JPQCD and 5-ASA. Comparing with the DSS group, the mRNA level of IL-1ß, IL-8, TNF-α and NF-κB was significantly reduced by 5-ASA and JPQCD. The difference between JPQCD and 5-ASA groups was not statistically significant (P > 0.05). Comparing with the DSS group, due to using JPQCD and 5-ASA, significant suppression of activation in DSS-induced NF-κB and increased phosphorylation of IκB in mice with experimental colitis occurred (P < 0.05). The difference between the JPQCD group and the 5-ASA group was not statistically significant (P > 0.05). CONCLUSION: Activation of the NF-κB signaling pathway is inhibited by JPQCD, which shows the potential mechanism by which JPQCD treats UC.

De Novo Sequencing and Transcriptome Analysis of Pleurotus eryngii subsp. tuoliensis (Bailinggu) Mycelia in Response to Cold Stimulation.

Cold stimulation of Bailinggu's mycelia is the main factor that triggers primordia initiation for successful production of fruiting bodies under commercial cultivation. Yet, the molecular-level mechanisms involved in mycelia response to cold stimulation are still unclear. Here, we performed comparative transcriptomic analysis using RNA-Seq technology to better understand the gene expression regulation during different temporal stages of cold stimulation in Bailinggu. A total of 21,558 Bailinggu mycelia unigenes were de novo assembled and annotated from four libraries (control at 25 °C, plus cold stimulation treatments at -3 °C for a duration of 1-2 days, 5-6 days, and 9-10 days). GO and KEGG pathway analysis indicated that functional groups of differentially expressed unigenes associated with cell wall and membrane stabilization, calcium signaling and mitogen-activated protein kinases (MAPK) pathways, and soluble sugars and protein biosynthesis and metabolism pathways play a vital role in Bailinggu's response to cold stimulation. Six hundred and seven potential EST-based SSRs loci were identified in these unigenes, and 100 EST-SSR primers were randomly selected for validation. The overall polymorphism rate was 92% by using 10 wild strains of Bailinggu. Therefore, these results can serve as a valuable resource for a better understanding of the molecular mechanisms associated with Bailinggu's response to cold stimulation.