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 Δku80 strain for efficient gene targeting. Dikaryons of P. ostreatus Δmsh4×Δmsh4 or Δmer3×Δmer3 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 Δmsh4×Δmsh4 dikaryons versus the P. ostreatus msh4+×Δmsh4 dikaryons, and 1/82.6 in the P. ostreatus Δmer3×Δmer3 dikaryons versus the P. ostreatus mer3+×Δmer3 dikaryons. To demonstrate the effects of ku80 disruption, P. ostreatus Δku80×Δku80 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.