iTRAQ-Based Quantitative Proteomic Analysis Reveals Proteomic Changes in Mycelium of Pleurotus ostreatus in Response to Heat Stress and Subsequent Recovery.

High temperature is a key limiting factor for mycelium growth and development in Pleurotus ostreatus. Thermotolerance includes the direct response to heat stress and the ability to recover from heat stress. To better understand the mechanism of thermotolerance in P. ostreatus, we used morphological and physiological analysis combined with an iTRAQ-based proteomics analysis of P. ostreatus subjected to 40°C for 48 h followed by recovery at 25°C for 3 days. High temperature increased the concentrations of thiobarbituric acid reactive substances (TBARS) indicating that the mycelium of P. ostreatus were damaged by heat stress. However, these physiological changes rapidly returned to control levels during the subsequent recovery phase from heat stress. In comparison to unstressed controls, a total of 204 proteins were changed during heat stress and/or the recovery phase. Wherein, there were 47 proteins that responded to both stress and recovery conditions, whereas 84 and 73 proteins were responsive to only heat stress or recovery conditions, respectively. Furthermore, quantitative real-time PCR (qRT-PCR) confirmed differential expression of nine candidate genes revealed that some of the proteins, such as a mitogen-activated protein kinase (MAPK), phenylalanine ammonia-lyase (PAL), and heat shock protein (HSP), were also regulated by heat stress at the level of transcription. These differentially expressed proteins (DEPs) in mycelium of P. ostreatus under heat stress were from 13 biological processes. Moreover, protein-protein interaction analysis revealed that proteins involved in carbohydrate and energy metabolism, signal transduction, and proteins metabolism could be assigned to three heat stress response networks. On the basis of these findings, we proposed that effective regulatory protein expression related to MAPK-pathway, antioxidant enzymes, HSPs, and other stress response proteins, and glycolysis play important roles in enhancing P. ostreatus adaptation to and recovery from heat stress. Of note, this study provides useful information for understanding the thermotolerance mechanism for basidiomycetes.

Genome-Wide Characterization and Expression Analyses of Pleurotus ostreatus MYB Transcription Factors during Developmental Stages and under Heat Stress Based on de novo Sequenced Genome.

Pleurotus ostreatus is a commercially grown mushroom species in China. However, studies on the mechanisms of the fruiting body development and stress response of P. ostreatus are still at a primary stage. In this study, we report the entire genome sequence of P. ostreatus CCMSSC03989. Then, we performed comprehensive genome-wide characterization and expression analysis of the MYB transcription factor family during a series of developmental stages and under the condition of heat stress. A 34.76 Mb genome was obtained through next-generation sequencing (NGS) and Bionano optical mapping approaches. The genome has a scaffold N50 of 1.1 Mb and contains 10.11% repeats, and 10,936 gene models were predicted. A total of 20 MYB genes (PoMYB) were identified across the genome, and the full-length open reading frames were isolated. The PoMYBs were classified into 1 repeat (1R), 2R, and 3R-MYB groups according to their MYB domain repeat numbers, and 3R-MYBs possessed relatively more introns than 1R and 2R-MYBs. Based on phylogenetic analysis, the PoMYBs were divided into four groups and showed close relationships with the MYB genes of plants and fungi. RNA-sequencing (RNA-Seq) and quantitative PCR (qPCR) analyses revealed that PoMYB expression showed stage-specific patterns in reproductive stages and could be induced by heat stress. The P. ostreatus draft genome will promote genome-wide analysis, and our study of PoMYBs will promote further functional analysis of MYB genes in mushrooms.

A genetic linkage map of Pleurotus tuoliensis integrated with physical mapping of the de novo sequenced genome and the mating type loci.

BACKGROUND: Pleurotus tuoliensis (Bailinggu) is a commercially cultivated mushroom species with an increasing popularity in China and other Asian countries. Commercial profits are now low, mainly due to a low yield, long cultivation period and sensitivity to diseases. Breeding efforts are thus required to improve agronomical important traits. Developing saturated genetic linkage and physical maps is a start for applying genetic and molecular approaches to accelerate the precise breeding programs. RESULTS: Here we present a genetic linkage map for P. tuoliensis constructed by using 115 haploid monokaryons derived from a hybrid strain H6. One thousand one hundred and eighty-two SNP markers developed by 2b-RAD (type IIB restriction-site associated DNA) approach were mapped to 12 linkage groups. The map covers 1073 cM with an average marker spacing of 1.0 cM. The genome of P. tuoliensis was de novo sequenced as 40.8 Mb and consisted of 500 scaffolds (>500 bp), which showed a high level of colinearity to the genome of P. eryngii var. eryngii. A total of 97.4% SNP markers (1151) were physically localized on 78 scaffolds, and the physical length of these anchored scaffolds were 33.9 Mb representing 83.1% of the whole genome. Mating type loci A and B were mapped on separate linkage groups and identified physically on the assembled genomes. Five putative pheromone receptors and two putative pheromone precursors were identified for the mating type B locus. CONCLUSIONS: This study reported a first genetic linkage map integrated with physical mapping of the de novo sequenced genome and the mating type loci of an important cultivated mushroom in China, P. tuoliensis. The de novo sequenced and annotated genome, assembled using a 2b-RAD generated linkage map, provides a basis for marker-assisted breeding of this economic important mushroom species.

Identification and Characterization of Small Noncoding RNAs in Genome Sequences of the Edible Fungus Pleurotus ostreatus.

Noncoding RNAs (ncRNAs) have been identified in many fungi. However, no genome-scale identification of ncRNAs has been inventoried for basidiomycetes. In this research, we detected 254 small noncoding RNAs (sncRNAs) in a genome assembly of an isolate (CCEF00389) of Pleurotus ostreatus, which is a widely cultivated edible basidiomycetous fungus worldwide. The identified sncRNAs include snRNAs, snoRNAs, tRNAs, and miRNAs. SnRNA U1 was not found in CCEF00389 genome assembly and some other basidiomycetous genomes by BLASTn. This implies that if snRNA U1 of basidiomycetes exists, it has a sequence that varies significantly from other organisms. By analyzing the distribution of sncRNA loci, we found that snRNAs and most tRNAs (88.6%) were located in pseudo-UTR regions, while miRNAs are commonly found in introns. To analyze the evolutionary conservation of the sncRNAs in P. ostreatus, we aligned all 254 sncRNAs to the genome assemblies of some other Agaricomycotina fungi. The results suggest that most sncRNAs (77.56%) were highly conserved in P. ostreatus, and 20% were conserved in Agaricomycotina fungi. These findings indicate that most sncRNAs of P. ostreatus were not conserved across Agaricomycotina fungi.

Genome-wide functional analysis of SSR for an edible mushroom Pleurotus ostreatus.

Simple sequence repeats (SSRs) play specific roles in many biological activities. In this paper, we focused on SSRs in the genome of Pleurotus ostreatus, which is a widely cultivated edible mushroom. The distribution curves of SSRs and exons are opposite throughout the genome, which means that SSRs are mostly located in non-coding regions. A comparative analysis of nine fungi suggests that Agaricomycotina fungi have similar SSR distributions. Functional enrichment analysis on the SSR-containing gene set uncovers enriched functions about environmental interactions and important cellular functions for life. Trinucleotide SSRs account for an extremely high fraction of all SSRs, and in exonic regions, they are equivalent to inserting repeating amino acids (RAAs) into the protein sequences. The RAA indel could partly explain some enriched functions of the genes they modify. Agaricomycotina fungi have similar distributions of RAAs, indicating that this may be a potential common mechanism for some specific functions.

Genetic variability and population structure of the mushroom Pleurotus eryngii var. tuoliensis.

The genetic diversity of 123 wild strains of Pleurotus eryngii var. tuoliensis, which were collected from nine geographical locations in Yumin, Tuoli, and Qinghe counties in the Xinjiang Autonomous Region of China, was analysed using two molecular marker systems (inter-simple sequence repeat and start codon targeted). At the variety level, the percentage of polymorphic loci and Nei's gene diversity index for P. eryngii var. tuoliensis was 96.32% and 0.238, respectively. At the population level, Nei's gene diversity index ranged from 0.149 to 0.218 with an average of 0.186, and Shannon's information index ranged from 0.213 to 0.339 with an average of 0.284. These results revealed the abundant genetic variability in the wild resources of P. eryngii var. tuoliensis. Nei's gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 1.794) was inferred as a major reason for the extent of genetic differentiation of P. eryngii var. tuoliensis. The results of Mantel tests showed that the genetic distance among geographical populations of P. eryngii var. tuoliensis was positively correlated with the geographical distance and the longitudinal distances (rGo = 0.789 and rLn = 0.873, respectively), which indicates that geographical isolation is an important factor for the observed genetic differentiation. Nine geographical populations of P. eryngii var. tuoliensis were divided into three groups according to their geographical origins, which revealed that the genetic diversity was closely related to the geographical distribution of this wild fungus.