Insights into the genomic evolution and the alkali tolerance mechanisms of Agaricus sinodeliciosus by comparative genomic and transcriptomic analyses.

Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the 'starch and sucrose metabolism', 'biosynthesis of amino acids' and 'phenylpropanoid biosynthesis' pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus.

Construction of a heat-resistant strain of Lentinus edodes by fungal Hsp20 protein overexpression and genetic transformation.

The shiitake mushroom (Lentinus edodes) is the second most popular edible mushroom globally due to its rich nutritional value and health benefits associated with consumption. However, the characteristics of growing at low temperatures limit the area and time of its cultivating. We selected a low-temperature cultivar as the original strain. We proposed to construct a heat-shock protein expression vector to achieve genetic transformation in this low-temperature strain to improve the survivability of the strain against the heat-shock response. In this study, an overexpression vector pEHg-gdp-hsp20 for the heat shock protein 20 gene of A. bisporus was constructed using a homologous recombination method. This vector was transferred into dikaryotic and monokaryotic mycelia by the Agrobacterium tumefaciens-method. The integration of hygb and hsp20 into the genome of L. edodes mycelia was verified by growth experiments on resistant plates and PCR analysis. The expression of the reporter gene mgfp5 was verified by fluorescence microscopy analysis and statistically resulted in 18.52 and 26.39% positivity for dikaryon, and monokaryon, respectively. Real-time PCR analysis showed that the expression of the hsp20 gene was more than 10-fold up-regulated in the three transformants; the mycelia of the three overexpression transformants could resume growth after 24 h heat treatment at 40°C, but the mycelia of the starting strain L087 could not recover growth at 25°C indicating that strains that successfully expressed hsp20 had greater overall recovery after heat shock. According to the study, A. bisporus hsp20 gene overexpression effectively improves the defensive capability of low-temperature mushroom strains against heat shock, laying the foundation for breeding heat-resistant high-quality transgenic shiitake mushrooms.

Four new species of Agaricus subgenus Spissicaules from China.

Agaricus subgenus Spissicaules is widely distributed in the world. In this study, 114 specimens were included in multigene phylogenetic analyses that allowed a better circumscription of the four sections in A. subg. Spissicaules. Three new species from China, A. brunneovariabilis, A. beijingensis, and A. planiceps, are described and placed in different sections. The invalidly described A. catenariocystidiosus is validated here as an additional new species. Comparisons between the sequences of the three closely related species A. thiersii, A. parasubrutilescens, and A. linzhiensis support the distinction between these three species despite the paraphyly of A. linzhiensis, which results from its high intraspecific variability with numerous heteromorphisms.

A revision of Agaricus section Arvenses with nine new species from China.

Agaricus sect. Arvenses includes numerous species that are potential candidates for cultivation, and some have high nutritional and medicinal interests. Between 2012 and 2017, 147 specimens of A. sect. Arvenses were collected in China. For this study, nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and translation elongation factor 1-alpha (tef1) sequences were used to assess species boundaries of these samples from China. Combined with morphological examination, we recognize 22 species of A. sect. Arvenses from China, of which 12 are known species, one is new record for China, and nine are proposed as new.

Offspring analysis using two cleaved amplified polymorphic sequence (CAPS) markers reveals amphithallism in the edible mushroom Agaricus sinodeliciosus.

Agaricus sinodeliciosus is an edible wild mushroom known in northwest China. It belongs to Agaricus section Bivelares that includes several popular cultivated species, such as A. bisporus, the button mushroom. The life cycle of the latter species has been described as amphithallic because both homokaryotic (n) and heterokaryotic (n+n) spores are produced that lead to heterothallic and pseudohomothallic life cycles, respectively. The type of life cycle can impact population structures and breeding strategies. The main objective of this study was to identify the different categories of spores produced by A. sinodeliciosus. Using either a morphological approach based on the number of sterigmata per basidium or a genetic approach based on the genotypes of the progeny at two loci, the proportion of heterokaryotic spores was estimated at 6% and 15%, respectively. Two codominant markers were chosen from the mitochondrial intermediate peptidase gene (MIP) and the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. Genotypic analysis and mating tests confirmed that in A. sinodeliciosus, MIP is centromere-linked and tightly linked to the mating type locus as in A. bisporus and A. subrufescens. We conclude that A. sinodeliciosus has a unifactorial system of sexual incompatibility and an amphithallic life cycle that is predominantly heterothallic, and that its pseudohomothallism follows a nonrandom model with nonsister postmeiotic nuclei paired in the same spore, which give rise to a potentially fertile heterokaryon. This method of using two informative markers is reliable not only in selecting the homokaryotic offspring but also in classifying the homokaryons in two breeding stocks according to their mating type alleles.