Molecular breeding of sporeless strains of Pleurotus ostreatus using a non-homologous DNA end-joining defective strain

Gene targeting is useful to isolate strains with mutations in a gene of interest for efficient breeding. In this study, we generated msh4 or mer3 single-gene disruptant monokaryons using a Pleurotus ostreatus Delta ku80 strain for efficient gene targeting. Dikaryons of P. ostreatus Delta msh4x Delta msh4 or Delta mer3x Delta mer3 were isolated via backcrosses, and the number of basidiospores produced was measured. The number of basidiospores fell by an average 1/13.7 in the P. ostreatus Delta msh4x Delta msh4 dikaryons versus the P. ostreatus msh4(+)x Delta msh4 dikaryons, and 1/82.6 in the P. ostreatus Delta mer3x Delta mer3 dikaryons versus the P. ostreatus mer3(+)x Delta mer3 dikaryons. To demonstrate the effects of ku80 disruption, P. ostreatus Delta ku80x Delta ku80 dikaryon strains were isolated and no significant effects on basidiospore production were observed. Fluorescence microscopy showed meiotic progression was arrested during prophase I in the msh4 or mer3 disruptants. To our knowledge, this is the first report on molecular breeding of sporeless strains in cultivated mushrooms using an efficient method for targeted gene disruption.