Paramarasmius mesosporus is a wild edible mushroom with a strong aroma and high culinary value, capable of growing at temperatures up to 36 °C. However, its domestication system and the genetic basis of its sexual reproduction remain largely unexplored. In this study, fresh fruiting bodies of P. mesosporus were collected from the rhizosphere of Imperata spp. in saline–alkali land in Hebei Province, China. The species was successfully domesticated for the first time, and fruiting bodies were harvested 7–10 days after spawning. A chromosome-level genome of the monokaryotic strain ‘Q2’ was assembled using whole-genome sequencing, transcriptome analysis, and Hi-C technology, with a total size of 46.57 Mb that was anchored onto 11 pseudochromosomes at a mounting rate of 95.18%. The mating-type system was identified as a typical tetrapolar heterothallic type, with the MAT A and MAT B loci located on chromosomes 2 and 10, respectively. The MAT A locus encodes HD1, HD2, MIP, and β-fg, among which tr-HD1 was found to be pseudogenized due to domain truncation. The MAT B locus comprises seven pheromone receptor genes and seven pheromone precursor genes arranged in an interspersed pattern. Based on these genomic features, a preliminary MAPK signaling pathway model was proposed to elucidate the molecular regulation of sexual reproduction. This study achieved the first artificial domestication and cultivation of P. mesosporus. systematically elucidated genomic characteristics and mating-type molecular mechanisms of this species were systematically elucidated, providing a theoretical foundation for hybrid breeding, molecular marker-assisted selection, and genetic improvement. These findings hold significant scientific and applied value for germplasm innovation and industrial development of this rare edible mushroom.

Stropharia rugosoannulata is a widely cultivated edible fungus with high economic and nutritional value. It is a tetrapolar heterothallic basidiomycete. The development of single nucleotide polymorphism (SNP) markers for mating-type identification holds considerable promise for enhancing breeding efficiency. In our study, one group of test crosses and three-round mating experiments from one parental strain were conducted in order to ascertain the mating type in this species. Segregation distortion in mating types was observed after single-spore isolation, which was deviated from Mendelian inheritance. The monokaryotic strain Q25 was derived from the dikaryotic mycelium S1 of S. rugosoannulata. The genome map of strain Q25 with 48.27 Mb and 14 chromosomes was constructed using genomic, transcriptomic, and high-throughput chromosome conformation capture (Hi-C) sequencing technologies. The locations of mating-type loci were identified using genomic annotation. The mating-type A locus is located in chromosome 1, with the gene sequence of β-fg, HD2, HD1, and MIP. The mating-type B locus is located in chromosome 12. It contains five pheromone receptors and five pheromone precursor genes. Two pairs of highly specific and stable primers were designed based on SNP loci in A and B mating types. A1, A2, B1, and B2 alleles were precisely distinguished with these primers, which were subsequently applied in cultivation experiments. This study lays a foundation for the precise breeding of S. rugosoannulata.