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Quantitative real-time PCR (qRT-PCR) is widely used to detect gene expression due to its high sensitivity, high throughput, and convenience. The accurate choice of reference genes is required for normalization of gene expression in qRT-PCR analysis. In order to identify the optimal candidates for gene expression analysis using qRT-PCR in Agaricus blazei, we studied the potential reference genes in this economically important edible fungus. In this study, transcriptome datasets were used as source for identification of candidate reference genes. And 27 potential reference genes including 21 newly stable genes, three classical housekeeping genes, and homologous genes of three ideal reference genes in Volvariella volvacea, were screened based on transcriptome datasets of A. blazei and previous studies. The expression stability of these genes was investigated by qRT-PCR analysis and further evaluated by four software packages, geNorm, NormFinder, BestKeeper, and RefFinder. Among these candidates, α-TUB (Tubulin alpha) and Cox5a (COX5A subunit VA of cytochrome c oxidase) were revealed as the most stable in fruit body, and suitable for 5 different developmental stages. α-TUB and ATP3 (ATP3 gamma subunit of the F1 sector of mitochondrial F1F0 ATP synthase) showed the most stable expression in stipe tissues and, Uqcrc (core subunit of the ubiquinol-cytochrome c reductase complex) and PUP3 (20S proteasome subunit beta 3) performed well in pileus tissues during the process of A. blazei development, while GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was among the least stable genes in all sample sets. Finally, the Ableln3 (homology of eln3 gene of Coprinus cinereus) was adopted to validate the reliability of these stable and unstable reference genes, indicating that the use of unsuitable reference genes as internal controls could change the target gene’s expression pattern. This study can provide guidance for choosing reference genes for analyzing the expression pattern of target genes and facilitate the functional genomic investigation on fruit body formation and development, as well as stipe elongation and pileus expansion in A. blazei.

Agaricus bisporus is widely used on the world market. The easy browning of mushroom surface is one of the most intuitive factors affecting consumer purchase. A certain cognition on browning mechanism have been made after years of research. At present, people slow down the browning of mushrooms mainly by improving preservation methods. In addition, breeding is also a reliable way. In the production practice, we have identified some browning resistant varieties,and we selected a browning-resistant variety to compare with ordinary variety to reveal the resistance mechanism. Using transcriptomics and metabolomics, the differences in gene expression and metabolite levels were revealed, respectively. The results showed that differentially expressed genes (DEGs) like AbPPO4, AbPPO3 and AbPPO2 were differently expressed and these DEGs involved in many pathways that related to browning. The expression of AbPPO expression play an important role in the browning of A. bisporus and multiple PPO family members are involved in the regulation of browning. However, the resistance to browning cannot be judged only by the expression level of AbPPOs. Formetabolomics, most of the different metabolites were organic acids. These organic acids had a higher level in anti-browning (BT) than easy-browning varieties (BS), although the profile was very heterogeneous. On the contrary, the content of trehalose in BS was significantly higher than that in BT. Higher organic acids decreased pH and further inhibited PPO activity. In addition, the BS had a higher content of trehalose, which might play roles in maintain the activity of PPO. The difference of browning resistance between BS and BT is mainly due to the differential regulation mechanism of PPO.

Agaricus bisporus is the most widely cultivated edible mushroom in the world. Strain quality has an important influence on the yield of A. bisporus, with strains that exhibit aging being a common problem during cultivation. However, little is known about the aging mechanisms of A. bisporus strain. In this study, the normal A. bisporus As2796 strain was compared to the aging A. bisporus As2796Y strain (which was previously discovered during cultivation). In the aging As2796Y mycelia, the mycelial growth rate and fruiting body yield were decreased and the chitin level and cell wall thickness were increased. Additionally, intracellular vacuoles increased, there was cytoplasmic shrinkage, and the sterol level which stabilizes the cell membrane decreased, which led to cytoplasmic outflow and the exudation of a large amount of yellow water from the mycelia. Additionally, there was increased electrolyte leakage. Furthermore, gas chromatography-mass spectrometry (GC/MS) was used to profile the metabolic changes in the aging As2796Y mycelia compared to the normal As2796 mycelia. A total of 52 differential metabolites were identified (75% were downregulated and 25% were upregulated in As2796Y). The reduction of many metabolites decreased the mycelial viability and the ability to maintain cell stability. Overall, this study is the first to report on the morphologic and metabolic changes in aged A. bisporus mycelia, which will aid future research on the mechanisms underlying A. bisporus mycelial aging.Key points• Aging of Agaricus bisporus strains will greatly reduce the fruiting body yield.• Aging of Agaricus bisporus strains can significantly change the cell structure of mycelia.• Many metabolites in the mycelium of aging spawn As2796Y significantly changed.

Agaricus blazei, a type of edible straw-rotting mushroom with somewhat sweet taste and fragrance of almonds, has attracted considerable scientific and practical attention. High-throughput Illumina PE150 and PacBio RSII platform were employed to generate a genomic sequence. De novo assembly generated 36 contigs with 38,686,133 bp in size, containing 10,119 putative predicted genes. Additionally, we also studied transcriptional regulation of the mycelia and the primordia for exploration of genes involved in fruiting body formation. Expression profiling analysis revealed that 2,164 genes were upregulated in mycelia and 1,557 in primordia. Functional enrichment showed that differentially expressed genes associated with response to stress, ribosome biogenesis, arginine biosynthesis, and steroid biosynthesis pathway were more active in fruiting body. The genome and transcriptome analysis of A. blazei provide valuable sequence resources and contribute to our understanding of genes related to the biosynthesis pathway of polysaccharide and benzaldehyde, as well as the fruiting body formation.