Resolving the Complex Genetic Basis of Phenotypic Variation and Variability of Cellular Growth

Abstract

In all organisms, the majority of traits vary continuously between individuals. Explaining the genetic basis of quantitative trait variation requires comprehensively accounting for genetic and nongenetic factors as well as their interactions. The growth of microbial cells can be characterized by a lag duration, an exponential growth phase, and a stationary phase. Parameters that characterize these growth phases can vary among genotypes (phenotypic variation), environmental conditions (phenotypic plasticity), and among isogenic cells in a given environment (phenotypic variability). We used a high-throughput microscopy assay to map genetic loci determining variation in lag duration and exponential growth rate in growth rate-limiting and nonlimiting glucose concentrations, using segregants from a cross of two natural isolates of the budding yeast, Saccharomyces cerevisiae We find that some quantitative trait loci (QTL) are common between traits and environments whereas some are unique, exhibiting gene-by-environment interactions. Furthermore, whereas variation in the central tendency of growth rate or lag duration is explained by many additive loci, differences in phenotypic variability are primarily the result of genetic interactions. We used bulk segregant mapping to increase QTL resolution by performing whole-genome sequencing of complex mixtures of an advanced intercross mapping population grown in selective conditions using glucose-limited chemostats. We find that sequence variation in the high-affinity glucose transporter HXT7 contributes to variation in growth rate and lag duration. Allele replacements of the entire locus, as well as of a single polymorphic amino acid, reveal that the effect of variation in HXT7 depends on genetic, and allelic, background. Amplifications of HXT7 are frequently selected in experimental evolution in glucose-limited environments, but we find that HXT7 amplifications result in antagonistic pleiotropy that is absent in naturally occurring variants of HXT7 Our study highlights the complex nature of the genotype-to-phenotype map within and between environments.

Keywords: HXT7; bulk segregant mapping; microcolony; quantitative trait locus; yeast.

Figure 1

Multiple QTL underlie phenotypic variation and variability of cell growth. Additive QTL identified for all analyzed traits are depicted. Chromosome size and QTL position correspond to genetic distance (in cM). Colors depict estimated effect sizes. Different shapes are used to distinguish distinct QTL on the same chromosome. Positive effects (blue) correspond to oak alleles that increase growth rate traits or decrease lag duration traits, whereas negative effects (red) correspond to vineyard alleles that increase growth rate traits or decrease lag duration traits. All loci were significant at α = 0.05 except those shown in gray, which were significant at α = 0.1.

Figure 2

Genetic interactions contribute to phenotypic variation and variability of cell growth. Significant genetic interactions identified for phenotypic variation and variability traits are shown as lines connecting loci. Chromosome size and QTL position correspond to genetic distance (in cM). Line widths correspond to the percent of variance explained when only the two interacting loci are modeled. Interactions underlying heritable variation in central tendency (pink) and heritable variation in variability (green) were identified. Different shapes distinguish distinct loci on the same chromosome and correspond to shapes in Figure 1. Interactions were significant at α = 0.05 except those indicated by dashed lines, which were significant at α = 0.1.

Figure 3

A sign epistatic interaction underlies heritable variation in growth rate variability. (A) LOD scores corresponding to additive (two-QTL) or full (two-QTL and interaction) models for each combination of chromosome I and X positions for linkage to phenotypic variability in growth rate measured in limiting glucose. (B) Average growth rate variability in the limiting glucose condition for the four genotype combinations corresponding to the two markers with the maximum LOD score difference shown in (A). Error bars represent SEs.

Figure 4

Variance in phenotypic variation is mainly explained by additive QTL whereas variance in phenotypic variability is mainly explained by genetic interactions. Percent of total trait variance explained per trait by a model comprising all identified additive QTL (black) or comprising all identified additive QTL and genetic interactions (gray) is shown.

Figure 5

Increased QTL resolution due to increased recombination in an advanced intercross population. (A) LOD score profiles for all of chromosome IV obtained by interval mapping using F2 segregants (dark purple) or sequencing under selection of an advanced intercross population (orange). For the interval mapping profile, genetic distances were converted to physical distances based on marker positions. The inset shows LOD profiles for a 15-kb region (gray region in main plot) centered on the peak LOD score and the corresponding physical position of genes within the QTL. (B) LOD score profiles for a region of chromosome VIII analyzed by sequencing under selection of a F2 pool (light purple) or using an advanced intercross population (orange). MULTIPOOL parameters for depicted data are n = 1000 and r = 1000.

Figure 6

Background-dependent effects on HXT7 contribute to cell growth rate variation. Distributions of (A) mean growth rate and (B) median lag duration for allele replacement strains grown in limiting glucose. P-values are for two sample t-tests (n = 12 for each analyzed strain). The diploid strain genotype and the genotype at the HXT7 locus are of either oak (blue) or vineyard (red) parental origin. Single-amino acid allele replacements within HXT7 are depicted as mosaics of oak and vineyard genotypes at the HXT7 locus.