Efficient selection of hybrids by protoplast fusion using drug resistance markers and reporter genes in Saccharomyces cerevisiae.

We have developed a selection system for hybrids by protoplast fusion using dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A, and reporter genes, ADH1p-PHO5-ADH1t and CLN2p-CYC1-lacZ, in Saccharomyces cerevisiae. To examine the effectiveness of this system, plasmids with each marker and reporter gene were introduced into auxotrophic sake yeasts. From the resulting transformants, eight colonies were screened by protoplast fusion in combination with the drug resistance markers and the reporter genes. Among them, seven strains were judged as hybrids between parental strains by analysis of growth on a minimal medium. This selection system was applied to wine yeasts having no genetic markers. Six strains were regarded as hybrids between parental strains by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene and by karyotype analysis using a contour-clamped homogeneous electric field (CHEF). We propose that the plotoplast fusion using dominant selective geneticin- and aureobasidin A-resistance markers and reporter genes is useful for the selection of hybrids from wine yeasts, which are homothallic and have low sporulation ability.