To improve our understanding of the selfing of G. lingzhi basidiospore monokaryons and increase the efficiency of breeding excellent strains, 52 basidiospore monokaryons were isolated from a commercial G. lingzhi strain (laboratory number P). A severe partial segregation was observed using the chi-square test, the growth rate of the monokaryotic strains was normally distributed, and colonies exhibited 5 forms. The genetic diversity of the monokaryotic strains was further demonstrated by intersimple sequence repeat (ISSR) analysis, and the similarity coefficient was in the range of 0.49–1, which was consistent with the genotype classification results. In total, 14 AxBx monokaryotic strains were randomly selected for selfing with the 1 AyBy strain when the similarity coefficient was 0.76, and a total of 14 offspring were obtained via selfing, all of which were incompatible with their parents. The traits of the selfing progenies were diverse. The mycelial growth rate, fruiting body yield, and polysaccharide, triterpene, and sterol contents were the main indices. According to the membership function value, 71.43% of the selfing progeny were super parent, and the A88 strain with the best comprehensive traits was selected. These findings prove that ISSR molecular marker-assisted breeding reduces blindness, greatly reduces workload, and improves work efficiency.
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