Genetic and Molecular Evidence of a Tetrapolar Mating System in the Edible Mushroom <i>Grifola frondosa</i>

<i>Grifola frondosa</i> is a valuable edible fungus with high nutritional and medicinal values. The mating systems of fungi not only offer practical strategies for breeding, but also have far-reaching effects on genetic variability. <i>Grifola frondosa</i> has been considered as a sexual species with a tetrapolar mating system based on little experimental data. In the present study, one group of test crosses and six groups of three-round mating experiments from two parental strains were conducted to determine the mating system in <i>G. frondosa</i>. A chi-squared test of the results of the test-cross mating experiments indicated that they satisfied Mendelian segregation, while a series of three-round mating experiments showed that Mendelian segregation was not satisfied, implying a segregation distortion phenomenon in <i>G. frondosa</i>. A genomic map of the <i>G. frondosa</i> strain, y59, grown from an LMCZ basidiospore, with 40.54 Mb and 12 chromosomes, was generated using genome, transcriptome and Hi-C sequencing technology. Based on the genomic annotation of <i>G. frondosa</i>, the mating-type loci A and B were located on chromosomes 1 and 11, respectively. The mating-type locus A coded for the β-fg protein, HD1, HD2 and MIP, in that order. The mating-type locus B consisted of six pheromone receptors (PRs) and five pheromone precursors (PPs) in a crossed order. Moreover, both HD and PR loci may have only one sublocus that determines the mating type in <i>G. frondosa</i>. The nonsynonymous SNP and indel mutations between the A₁B₁ and A₂B₂ mating-type strains and the reference genome of y59 only occurred on genes <i>HD2</i> and <i>PR1</i>/<i>2</i>, preliminarily confirming that the mating type of the y59 strain was A₁B₂ and not A₁B₁. Based on the genetic evidence and the more reliable molecular evidence, the results reveal that the mating system of <i>G. frondosa</i> is tetrapolar. This study has important implications for the genetics and hybrid breeding of <i>G. frondosa</i>.