Effects of mating-type ratio imbalance on the degeneration of subculture and preventative measures.

The rapid degeneration of strains during subculture represents a bottleneck problem that affects production stability. This study explored the mechanism underlying this degeneration in three production and three wild-type strains of , isolating single-conidium strains from each. The effects of subculturing on fructification in both original and single mating-type strains were compared. Changes in the ratio of the two mating types were analyzed in both original and degenerated strains. Based on these findings, the two mating strains were paired in different ratios to determine their effects on fruiting. The resulting five strains were heterokaryotic strains with both MAT1-1 and MAT1-2 mating-type genes. Strain jb-2 was a single mating type (MAT1-1) mutant strain that produced stable fruiting bodies but failed to produce ascospores. It was found that the loss of or imbalance in mating types was the main reason for the rapid degeneration of fruiting traits during subculture and that this occurred randomly in the MAT1-1 and MAT1-2 types. The strains differed significantly in their stability during subculture. Fruiting was stable in the single mating-type Jb-2 strain, and the eleventh-generation fruited normally. There were differences in yield between the production and wild strains after inoculation with spawn containing different proportions of mating types. The production strain was more stable when inoculated with strains with mating-type ratios of 1:9 to 9:1 without affecting the yield. However, the yield of the wild-type strain xf-1 was positively correlated with the proportion of the MAT1-2 type, while the other two strains showed no correlations. Subculturing single mating-type mycelia separately and mixing them before production effectively mitigated degeneration during subculture. For breeding, selecting strains containing both mating types, which are insensitive to the proportion of mating-type genes, enhanced stability in subculture and reduced the risk of mating-type loss. Direct breeding of specific single-mating type strains to induce fruiting is thus an effective breeding strategy.