Abstract: To screen for Pholiota nameko hybrid strains with potential breeding value, six closely related strains of the genus Pholiota were used as experimental materials. Pure mycelial cultures were obtained by the tissue isolation method, and molecular identification and phylogenetic analysis were performed based on ITS (internal transcribed spacer) sequences. In addition, specific primers targeting the A mating-type locus were used for PCR amplification to detect the mating-type genes of the parental strains and their monospore isolates. Subsequently, monospore isolates with different mating types were paired for cross-hybridization, and the resulting hybrid strains were preliminarily screened by antagonism tests. The results showed that the six tested strains were classified into three species. Specifically, strain G6 was identified as Pholiota nameko, strain G4 as Pholiota limonella, and strains G1, G2, G3, and G5 as Pholiota microspora, with certain phylogenetic differentiation observed among the tested strains. A total of six A mating-type factors, namely A1, A2, A3, A4, A5, and A6, were detected among the tested strains, and the mating-type genotypes of the six parental strains G1-G6 were A1A2, A2A4, A1A4, A3A5, A5A6, and A3A4, respectively. A total of 112 cross combinations were obtained through monospore × monospore hybridization, and 18 candidate hybrid strains with stable genetic traits were finally selected through antagonism testing.

Key words: Pholiota nameko, monospore isolates, mating type, molecular markers, directed hybridization breeding