Different sets of QTLs influence fitness variation in yeast

Abstract

Most of the phenotypes in nature are complex and are determined by many quantitative trait loci (QTLs). In this study we identify gene sets that contribute to one important complex trait: the ability of yeast cells to survive under alkali stress. We carried out an in-lab evolution (ILE) experiment, in which we grew yeast populations under increasing alkali stress to enrich for beneficial mutations. The populations acquired different sets of affecting alleles, showing that evolution can provide alternative solutions to the same challenge. We measured the contribution of each allele to the phenotype. The sum of the effects of the QTLs was larger than the difference between the ancestor phenotype and the evolved strains, suggesting epistatic interactions between the QTLs. In parallel, a clinical isolated strain was used to map natural QTLs affecting growth at high pH. In all, 17 candidate regions were found. Using a predictive algorithm based on the distances in protein-interaction networks, candidate genes were defined and validated by gene disruption. Many of the QTLs found by both methods are not directly implied in pH homeostasis but have more general, and often regulatory, roles.

Figure 1

General strategy for mapping quantitative trait loci (QTLs) affecting genetic variability. See text for details. (A) In-lab evolution. A strain with poor ability to grow under alkali stress was serially transferred to media of increasing pH. (B) Construction of congenic lines up to the eighth generation. (C) Serially transferred cultures accumulate beneficial mutations. After long-term selection at increasing pH levels, cells were isolated from low pH media and individual colonies were grown and tested for their ability to grow at high pH. The ancestor (BY4741) and colonies derived from individual selection lines are shown. (D) Map of QTLs affecting the MP phenotype. Schematic representation of the 16 chromosomes of S. cerevisiae (black circles represent centromeres). Regions that were inherited from the high MP parent in the congenic lines and were identified by hybridization to oligonucleotide arrays are marked in red. QTLs showing mutations in the in-lab-evolved strains are marked green, yellow and orange.

Figure 2

Several genetic networks were selected during the in-lab evolution. (A) Phenotype distribution among 210 spores derived from a cross between a selected line (A8.5) and its low MP ancestor. (B) Clones from populations A8.5 and C8.6 were crossed to the low MP ancestor. The 10-fold dilutions of diploid yeast cells were plated on YPD at pH 6 and high pH solid media. In each case, the hybrid had a phenotype different from that of each parent. The hybrids also show different MP phenotypes. (C) The hybrid between two selected lines shows a low MP phenotype, indicating that most of the mutations that occurred in these lines are recessive and affect different QTLs.

Figure 3

Validation of the effect of each mutation found in evolved lines. (A) Drop assay for reciprocal hemizygote pairs to test the relative effect of each allele. In each pair of strains one of the alleles (from the ancestor or evolved origin) was deleted. (B) Results of a drop assay for reciprocal hemizygote pairs derived from line A8.5. (C) A single SNP affects two genes that contribute to the ability to grow at high pH. Drop assay for reciprocal hemizygote pairs of NMD4 and its adjacent, divergently transcribed ORF YLR363w-A (of unknown function). Both genes contribute to the ability to grow under alkali stress.

Figure 4

Quantification of the effect of each mutation using allele swapping. In all, 14 ‘Allele swap’ strains were created by introducing, in the low MP ancestor background, a single mutated allele from a high MP evolved strain. Growth curves were obtained for three different cultures of each of these strains and the controls at pH 6.0 and pH 8.0. Each column represents the ratio between the growth rate of a given strain and the average growth rate of the ancestor strain at high pH (8.0). The three dots on each column represent the calculated ratios for each strain. The colors represent alleles identified in different parent lines. The pale bars in the background provide the ratio based on the average of the three repeats. The strains that improve significantly growth at high pH (P<0.05, FDR corrected) are bolded. Source data is available for this figure at www.nature.com/msb.

Figure 5

Genetic variability in the ability to grow at high pH. (A) Fitness measurement of strains GRA2 and BY4741. The doubling time (DT) of each strain was measured while growing on YPD pH 6 or pH 7.9 during logarithmic phase. (B) A cross between the low MP parent (BY4741) and the high MP parent (GRA2) results in hybrid vigor (heterosis). Serial 10-fold dilutions of diploid strains were plated on regular and high-pH media. (C) Relative fitness of the three strains.

Figure 6

The CTR3 gene contributes to the MP phenotype. (A) RT–PCR showing the differences in transcription levels of CTR3 in different strains. ACT1 served as a control. (B) The ability of the various strains to grow at regular or high pH media (10-fold serial dilutions). Overexpression of CTR3 allows growth at higher MP.