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The mass scattering of basidiospores during the cultivation of edible mushrooms causes serious problems, such as allergic reactions in workers. Sporulation-deficient (sporeless) cultivars would be very useful for preventing these issues. We aimed to identify the single-nucleotide polymorphism (SNP) that is responsible for the single dominant sporeless mutation of the Tamogitake 108Y2D mutant using next-generation sequencing (NGS) and TILLING technology and to develop an allele-specific PCR marker for sporeless breeding. By comparing th