18

Noda Institute for Scientific Research GRANT
2014 Research Grant
Outline of Research Result

Genetic analysis of the ethanol fermentation ability of yeast
using a next-generation DNA sequencer
Hitoshi Shimoi
Faculty of Agriculture, Iwate University

Research aims

SnpEff toolbox.

Sake yeast is a diploid yeast classified as Saccharomyces
cerevisiae. In sake brewing, it is well known to produce
sake with good aroma and flavor as well as a high concentration of ethanol. Identification of the genes involved inthese sake brewing characteristics is important from theviewpoints of scientific research and for application inyeast breeding. Quantitative trait locus (QTL) analysis is amethod used to identify genetic loci linked to a specificphenotype on the basis of phenotyping and genotyping ofmany meiotic segregants of a hybrid, and has been applied
to many organisms with sexual life cycles. The authors previously isolated haploid segregants from the hybrid diploidof a sake yeast haploid and a laboratory yeast, and reported
QTL analysis of their sake brewing characteristics using
microsatellite DNA1). However, the resolving power of thisstudy was not high because of the low density of the DNA
markers on the whole genome. In this study, the genotypesof a pool of superior (or inferior) haploid segregants withethanol fermentation ability were analyzed at a single nucleotide polymorphism (SNP) level, using a next-generation sequencing method, in order to analyze more precise
QTLs.

Results

Methods
The best 10 (18.5–17.3%) and worst 10 (10.2–5.8%)
haploid segregants in terms of ethanol concentration in sakebrewing were selected among 100 haploid segregants derived from the hybrid diploid of a sake yeast Kyokai no. 7(K7) haploid strain (K7H868) and a laboratory yeast haploid strain (X2180-1B)1). Each strain of the best 10 haploid
segregants was cultured separately and an equivalent quantity of each strain was pooled, following which genome
DNA preparation was carried out (the best 10 pool). Genome DNA was also prepared from a pool of the worst 10segregants and from the parental strains (K7H868 and
X2180-1B). DNA sequences of the samples were analyzed
using Illumina HiSeq 2500 with the paired-end method.
The reads obtained were mapped on the S. cerevisiae
S288C genome (a laboratory strain) with the BWA software, after removing low-quality reads. Extraction of SNPs
from the mapped reads was performed with the GATK toolkit, whereas annotation of the SNPs was done with theThe total of high-quality sequencing reads obtained fromthe best 10 pool and the worst 10 pool was 2,092,574,200
bases and 1,682,096,400 bases, with sequencing redundancies of 138 and 114, respectively. After mapping on the laboratory yeast genome, 71,618 SNPs that were different between K7H868 and X2180-1B were extracted. For theseSNPs, whether each read obtained from the best 10 pooland the worst 10 pool was originated from the sake yeast(K7H868) genome or the laboratory yeast (X2180-1B) genome was determined2). The SNP frequency was defined as
1 when all SNPs were originated from the sake yeast genome, and as 0 when all SNPs were originated from thelaboratory yeast genome (Fig. 1). A high SNP frequency ata given SNP position in the best 10 pool indicates that thesake yeast-type SNP is associated with higher ethanol fermentation ability, whereas a low SNP frequency indicatesthat the laboratory yeast-type SNP is associated with higherethanol fermentation ability. In the worst 10 pool, the reverse association would be seen between SNP frequency
and ethanol fermentation ability.
One of the regions with high SNP frequency in the best
10 pool and low SNP frequency in the worst 10 pool was at
the position of ~70 kb on chromosome VI (Fig. 1). This region contains RIM15, the mutation of which has beenshown by individual genetic analysis to be involved in ethanol fermentation ability. RIM15 is required for the acquisition of stress tolerance after entering the stationary phase.
Sake yeast K7 has, however, a loss-of-function mutation inRIM15. This rim15 mutation is thought to be one of the reasons for the high ethanol fermentation ability of K7 and related sake yeast strains, since the laboratory yeast strain
having the rim15 mutation showed higher ethanol fermentation ability than the parental RIM15 strain3).
On the contrary, a region with very low SNP frequency(0) in the best 10 pool and high SNP frequency in the worst
10 pool was observed at the position of ~350 kb on chromosome XIII. This suggests that sake yeast K7 contains amutation with a negative effect on ethanol fermentation
ability.

		

19Fig. 1. Genome-wide SNP frequency of the pooled segregants.
SNP frequency of each pool was plotted against the chromosome coordinate. Red points indicate SNP frequency of the best 10 pool and blue points indicate
SNP frequency of the worst 10 pool. When this value is 1, all SNP of this position are derived from the sake yeast. When this value is 0, all SNP of this position are derived from the laboratory yeast.

Conclusion

References

QTL analysis of the ethanol fermentation ability of sakeyeast was carried out using next-generation sequencing ofgenome DNA of the pooled segregants derived from thehybrid between the sake yeast and laboratory yeast strains.
This method is very effective for QTL analysis of industrial
yeast strains since SNP frequencies at a large number of
SNP positions can be simultaneously determined by using
only one sequencing run.

1)

Katou, T., Namise, M., Kitagaki, H., Akao, T., and Shimoi, H.
(2009) QTL mapping of sake brewing characteristics of yeast. J.
Biosci. Bioeng. 107: 383–393.
2) Pais, T. M., Foulquié-Moreno, M. R., and Thevelein, J. M. (2014)
QTL mapping by pooled-segregant whole-genome sequencing inyeast. Methods Mol. Biol. 1152: 251–266.
3) Watanabe, D., Araki, Y., Zhou, Y., Maeya, N., Akao, T., and Shimoi,H. (2012) A loss-of-function mutation in the PAS kinase Rim15p isrelated to defective quiescence entry and high fermentation rates in
Saccharomyces cerevisiae sake yeast strains. Appl. Environ. Microbiol. 78: 4008–4016.