Tetrad analysis of sake yeast and identification of an RFLP marker for the absence of phenolic off-flavour production

Mating is a promising breeding method for industrial yeast. Although sake yeast has a low spore-formation ability, segregants exhibiting a mating type have been isolated from sake yeast K7. Here, we constructed zygotes from a cross between those segregants and a laboratory yeast strain. Because most sake and brewing yeast strains are prototrophs, we developed a PCR-based method to confirm that mating had taken place based on genome sequencing data and differences in nucleotide sequences between the two parental strains. The mated strain, termed S. cerevisiae MITOY123, showed restored spore-formation ability, unlike most sake and brewing yeast strains. By using the mated yeast strain MITOY123, it was possible to carry out tetrad analysis for the trait of the absence of off-flavour due to phenolic products such as 4-vinylguiacol (4-VG) in sake yeast K7. This tetrad analysis indicated that a single genetic region around the gene PAD1 is responsible for the absence of phenolic off-flavour in sake yeast K7. In order to aid the breeding of sake and brewing yeast strains by mating, we also identified a restriction fragment length polymorphism (RFLP) marker for the absence of phenolic off-flavour production in strains derived from sake yeast K7. Collectively, our data show that it is possible to breed new sake and brewing yeast strains by mating and to test for the absence of phenolic off-flavour production in resultant strains easily by RFLP analysis.