Identification of novel causative genes determining the complex trait of high ethanol tolerance in yeast using pooled-segregant whole-genome sequence analysis

Abstract

High ethanol tolerance is an exquisite characteristic of the yeast Saccharomyces cerevisiae, which enables this microorganism to dominate in natural and industrial fermentations. Up to now, ethanol tolerance has only been analyzed in laboratory yeast strains with moderate ethanol tolerance. The genetic basis of the much higher ethanol tolerance in natural and industrial yeast strains is unknown. We have applied pooled-segregant whole-genome sequence analysis to map all quantitative trait loci (QTL) determining high ethanol tolerance. We crossed a highly ethanol-tolerant segregant of a Brazilian bioethanol production strain with a laboratory strain with moderate ethanol tolerance. Out of 5974 segregants, we pooled 136 segregants tolerant to at least 16% ethanol and 31 segregants tolerant to at least 17%. Scoring of SNPs using whole-genome sequence analysis of DNA from the two pools and parents revealed three major loci and additional minor loci. The latter were more pronounced or only present in the 17% pool compared to the 16% pool. In the locus with the strongest linkage, we identified three closely located genes affecting ethanol tolerance: MKT1, SWS2, and APJ1, with SWS2 being a negative allele located in between two positive alleles. SWS2 and APJ1 probably contained significant polymorphisms only outside the ORF, and lower expression of APJ1 may be linked to higher ethanol tolerance. This work has identified the first causative genes involved in high ethanol tolerance of yeast. It also reveals the strong potential of pooled-segregant sequence analysis using relatively small numbers of selected segregants for identifying QTL on a genome-wide scale.

Figure 1.

Ethanol tolerance of the Brazilian bioethanol production strain VR1 and its segregant VR1-5B. The ethanol tolerance of VR1 (diploid) and VR1-5B (haploid) was determined by scoring growth of tenfold dilutions on YP plates with different concentrations of ethanol. Both strains, as well as the heterozygous VR1-5B/BY4741 strain (diploid) showed a clearly higher ethanol tolerance than the control laboratory strains BY4741 (haploid) and BY (diploid), the latter of which was obtained by crossing BY4741 with BY4742.

Figure 2.

Genetic mapping of QTL involved in high ethanol tolerance by whole-genome sequence analysis. QTL were mapped by whole-genome sequence analysis of DNA extracted from a pool of 136 segregants tolerant to at least 16% ethanol (16% pool; green line) and from a pool of 31 segregants tolerant to at least 17% ethanol (17% pool; red line). The genomic DNA of the parents, VR1-5B and BY4741, and of the two pools, was sequenced and aligned to identify SNPs. The nucleotide frequency of quality-selected SNPs in the sequence of each pool was plotted against the chromosomal position. Significant deviations from the average of 0.5 indicate candidate QTL linked to high ethanol tolerance. Upward deviations indicate linkage to QTL in the ethanol-tolerant parent VR1-5B. The three major QTL on chromosomes V, X, and XIV are not significantly different between the two pools. However, in several instances, e.g., on chromosomes II, XII, and XV, minor loci can be identified, showing a significant difference between the two pools. These candidate QTL are more distinctive in the 17% pool compared to the 16% pool. The difference in SNP frequency between the two pools is certainly significant when the simultaneous confidence bands do not overlap.

Figure 3.

Detailed linkage statistics of QTL3, the locus with the strongest linkage to high ethanol tolerance. The table shows for each marker in the mapped QTL3 on chromosome XIV the position of the marker, the number of segregants in which the marker was scored, the association percentage, and the P-value. The association percentage represents the percentage of segregants with VR1-5B inheritance, i.e., the nucleotide from VR1-5B. The marker with the strongest linkage is shown in bold.

Figure 4.

Fine-mapping and identification of the causative genes in QTL3. (A) The 87-kb locus defined by SNP markers S67, S68, and S69 in QTL3 showed the lowest probability of random segregation in 101 highly ethanol-tolerant segregants. Further fine-mapping was achieved by scoring five additional markers within the 87-kb interval in the same segregants. Calculation of the P-values revealed the strongest linkage for a 16-kb locus defined by markers S68, S68-1, and S68-2. (B) The name and location of each ORF in the fine-mapped locus is shown as annotated in SGD (Cherry et al. 1997). The interval from nucleotide 466,599 to 485,809 was sequenced in VR1-5B and BY4741, which revealed 115 polymorphisms, of which part were in intergenic regions (numbers in parentheses). For the ORFs, only polymorphisms that change the amino acid sequence are indicated (amino acid in BY4741, followed by position in the protein and amino acid in VR1-5B). SAL1 has a frame shift mutation in BY4741 resulting in an earlier stop codon and truncation of the protein, which is assumed to be a loss-of-function gene product (Dimitrov et al. 2009). PMS1 has an insertion of four amino acids at position 417 in VR1-5B. The sequence of BY4741 in this interval is the same as that of S288c (Cherry et al. 1997), except for one nucleotide in SAL1 that causes an amino acid change at position 131 (valine in BY4741 and methionine in S288c and VR1-5B). (C) Reciprocal hemizygosity analysis. For the nine genes in the fine-mapped locus, two diploid strains were constructed in the VR1-5B/BY4741 hybrid background that carried either the VR1-5B (left) or BY4741 (right) allele from the gene. The rest of the genome was identical between the two hybrids. The reciprocal deletions were engineered in the haploid strains, after which the proper haploids were crossed to obtain the diploid hybrids. The ethanol tolerance of the diploid hybrids was determined by scoring the growth of twofold dilutions on 16% ethanol after 9 d. This revealed different contributions of the parental alleles from MKT1, SWS2, and APJ1 to high ethanol tolerance. The strain pairs were always spotted on the same plate. The results were assembled from different plates, thus slight differences in growth may be present between hybrid pairs that otherwise do not show differences in ethanol tolerance. Hence, only growth differences between strains within a hybrid pair are relevant. The growth of the wild-type diploid hybrid was similar to that of the hybrid pairs whose ethanol tolerance was unaltered.

Figure 5.

Effect of MKT1, SWS2, and APJ1 on ethanol tolerance. (A) The ethanol tolerance of BY4741 (inferior wild type) and the mkt1Δ, sws2Δ, and apj1Δ mutants thereof was determined by scoring growth of twofold dilutions on 10% ethanol after 8 d and 14% ethanol after 12 d. (B) The ethanol tolerance of VR1-5B (superior wild type) and the mkt1Δ, sws2Δ, and apj1Δ mutants thereof was determined by scoring growth of twofold dilutions on 10% ethanol after 6 d and 16% ethanol after 10 d. (C) The VR1-5B allele of MKT1 is beneficial for high ethanol tolerance. MKT1-BY and MKT1-VR, including 534 bp upstream and 344 bp downstream regions of the ORF, were cloned into the low-copy-number plasmid YCplac111 and expressed in BY4741 (BY1) and three segregants from VR1-5B/BY4741 that inherited the MKT1-BY allele (1D, 24A, and 32B). The ethanol tolerance was determined in twofold dilutions on different concentrations of ethanol.