Screening of heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium

A suitable screening method for heterozygous DNA markers in shiitake, Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments among the original strain (Hokken600:H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of 135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers tested. Furthermore, we tested whether the polymorphic fragments segregated into 2:2 among four strains isolated from a basidium. Most of the polymorphic fragments (about 97.8%) showed 2:2 segregation among the four strains. We concluded that the polymorphic fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to 2:2 among four meiotic strains (H600B-1, 2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from a dikaryon of shiitake (Hokken600: H600).