Molecular characterization of new natural hybrids of Saccharomyces cerevisiae and S. kudriavzevii in brewing

Abstract

We analyzed 24 beer strains from different origins by using PCR-restriction fragment length polymorphism analysis of different gene regions, and six new Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrid strains were found. This is the first time that the presence in brewing of this new type of hybrid has been demonstrated. From the comparative molecular analysis of these natural hybrids with respect to those described in wines, it can be concluded that these originated from at least two hybridization events and that some brewing hybrids share a common origin with wine hybrids. Finally, a reduction of the S. kudriavzevii fraction of the hybrid genomes was observed, but this reduction was found to vary among hybrids regardless of the source of isolation. The fact that 25% of the strains analyzed were discovered to be S. cerevisiae x S. kudriavzevii hybrids suggests that an important fraction of brewing strains classified as S. cerevisiae may correspond to hybrids, contributing to the complexity of Saccharomyces diversity in brewing environments. The present study raises new questions about the prevalence of these new hybrids in brewing as well as their contribution to the properties of the final product.

FIG. 1.

MP tree that minimizes the number of nucleotide substitutions required to connect the mitochondrial COX2 sequences from S. cerevisiae × S. kudriavzevii hybrids and type and reference strains of the Saccharomyces species. Brewing hybrids are included within squares, wine hybrids are indicated in italics, and the cider strain is underlined. The different COX2 haplotypes are given in bold. Numbers in italics located under the branches correspond to branch lengths given in nucleotide substitutions. Numbers at the nodes correspond to BVs (percent) obtained from 2,000 pseudoreplicates.

FIG. 2.

Chromosomal profiles exhibited by the brewing S. cerevisiae × S. kudriavzevii hybrids under analysis. Some wine S. cerevisiae × S. kudriavzevii hybrids, W27 and W46, the triple hybrid CID1, and representatives of the parental species, S. cerevisiae FY 1679 and S. kudriavzevii IFO 1802^T, are also included. Lane m corresponds to the standard marker strain S. cerevisiae YNN295 (Bio-Rad); chromosomal numbers corresponding to each band are indicated on the left.

FIG. 3.

Genotypes of the S. cerevisiae × S. kudriavzevii hybrids. Each square corresponds to a copy of each gene region according to its chromosome location, indicated at the left. White and black squares represent alleles of S. cerevisiae and S. kudriavzevii origin, respectively. Brewing and wine hybrids are indicated in bold and italics, respectively. The presence or absence of alleles coming from each parent species was determined by restriction analysis of the 35 gene regions amplified by PCR with general primers.

FIG. 4.

Minimum number of chromosomal rearrangements and restriction site changes to connect the different genotypes exhibited by the S. cerevisiae × S. kudriavzevii hybrids (Fig. 3; see also Table S••• in the supplemental material). Genotypes are represented by white and gray circles for wine and brewing hybrids, respectively. Rearrangements are indicated by arrows giving the direction of the irreversible change. Rearrangements were assumed to be caused by nonreciprocal recombination (rec) among homoeologous chromosomes (roman numbers) and whole chromosome losses (loss) of one of the parental chromosomes (kud, S. kudriavzevii). Restriction site changes can be reversible (gain/loss) and are represented by diamonds. The gene region and the restriction patterns involved are also indicated (for a description, see Tables S2 and S3 in the supplemental material). Dotted squares group genotypes of hybrids according to their mitochondrial COX2 haplotypes.