Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance

Abstract

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cant be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Toward this end we pioneered an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward phenotyping-by-sequencing. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening and sequencing identified dozens of loci at which variation between the species contributed to the thermotolerance trait. These hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, our results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

Temperature-Dependent Genetics of Thermotolerance Between Yeast Species

“…Examining our four focal thermotolerance genes, we detected an enrichment for low, negative Tajima's D at these loci across S. cerevisiae genomes (P = 0.0263; Supplementary Figure 3 and Supplementary Table 3A), reporting an excess of rare variants-as expected after a selective sweep, or under constraints from purifying selection (Biswas and Akey, 2006;Suzuki, 2010). We repeated this analysis using more comprehensive sets of hits from interspecies thermotolerance screens (Weiss et al, 2018;Abrams et al, 2021Abrams et al, , 2022, and detected strong signal for low, negative Tajima's D at these loci in vineyard/European S. cerevisiae in every case (Supplementary Tables 3B,C). Interestingly, ESP1 exhibited the most negative Tajima's D value among all thermotolerance genes in these analyses (Supplementary Table 3), dovetailing with the strong effect of variation at this gene in phenotypic analyses (Weiss et al, 2018; Figure 1).…”

“…For an initial study of the genetics of yeast species variation in temperature response, we chose to harness DBVPG1373, a homozygous diploid S. cerevisiae strain derived from an isolate from Dutch soil, and Z1, a homozygous diploid S. paradoxus strain derived from an isolate from an oak tree in England. We anticipated that detailed analyses using these strains, as representatives of their respective species, could accelerate the discovery of more general principles (Weiss et al, 2018;Abrams et al, 2021Abrams et al, , 2022. We developed an assay quantifying cell viability in a given liquid culture before and after incubation at a temperature of interest, and we implemented this approach for each species in turn.…”

“…To gain insight into past and current selective pressures on thermotolerance loci, we turned to a molecular-evolution approach. Previous work on Saccharomyces thermotolerance genes has emphasized sequence divergence between species (Weiss et al, 2018;Abrams et al, 2021Abrams et al, , 2022. For a complementary focus on population variation within S. cerevisiae, we analyzed the Tajima's D statistic, which formulates properties of sequence variants in a population into a test of how well the sequence fits the expectations under a neutral evolutionary model (Tajima, 1989;Biswas and Akey, 2006).…”

“…In previous work, a whole-genome mapping scheme identified housekeeping genes at which variation between S. cerevisiae and S. paradoxus impacts growth at the high end of the S. cerevisiae temperature range (Weiss et al, 2018). These loci exhibit striking sequence differences between the species, and conservation in S. cerevisiae, consistent with a history of positive selection on pro-thermotolerance alleles (Weiss et al, 2018;Abrams et al, 2021Abrams et al, , 2022. However, we have as yet little insight into the ecological dynamics by which this model trait evolved along the S. cerevisiae lineage.…”

“…Given that we have no access to genotypes representing ancient intermediates between this species and S. paradoxus, we designed a strategy to interrogate the genetics of extant strains, focusing on contributing genes of major effect. We surveyed gene-environment interactions by these thermotolerance loci across warm temperatures, complementing previous studies of interspecies variation at a single high temperature (Weiss et al, 2018;Abrams et al, 2022). And we investigated the frequency of variants at these loci with a population-genomic approach.…”