A simple genotyping method for <i>RIM15</i> gene polymorphisms in sake yeast strains

In the process of brewing sake, Saccharomyces cerevisiae produces alcohol and a wide range of flavor compounds. A previous study identified a sake yeast mutant with a single-nucleotide insertion in the RIM15 protein kinase gene (RIM15 with an insertion of adenine at position 5067: RIM15ins5067A). In this study, we developed primer sets (P1/P2) with engineered mismatches that introduce restriction sites for the restriction enzyme CspCI. This allowed us to create a simple and rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify Kyokai no. 7 (K7) group sake yeast strains carrying the RIM15ins5067A mutation. We validated the PCR-RFLP method in detecting the RIM15ins5067A mutation in the K7 group of yeast strains. This approach provides a straightforward, effective, and valuable tool for distinguishing specific yeasts used in sake production.