<p>Primer sets for mating type detection.</p>

Pleurotus ostreatus, a widely cultivated edible oyster mushroom, is an ecologically versatile species with applications in biotechnology, agriculture, and food production. It functions as a decomposer and in nutrient-limited conditions it enhances its survival by using a potent toxin to prey on nematodes. Its adaptability is further regulated by sexual reproduction, which follows a tetrapolar mating system governed by two unlinked, multiallelic loci, matA and matB. The two mating-compatible monokaryotic strains PC9 and PC15, derived from the parental dikaryon strain N001, exhibit significant physiological differences. PC9 grows robustly in laboratory conditions, whereas PC15 grows more slowly, making PC9 the preferred strain for research. To advance P. ostreatus as a genetic model, we characterized the mating-type (MAT) loci of both monokaryon strains and developed a congenic strain. We analyzed the MAT loci in multiple P. ostreatus strains, and identified 11 A and 12 B alleles among twelve haplotypes, confirming their multiallelic nature. Using 10 rounds of backcrossing, we introgressed the matA and matB loci from PC15 into the PC9 genetic background to generate the congenic strain PC9.15. After sequencing and assembling a high-quality and contiguous genome for PC9.15, we confirmed that the genomes of PC9.15 and PC9 are 99% similar, with the only major difference placed at the matA and matB loci.