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 cannot be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an interspecies hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here, we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. 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 identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these 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.

Keywords: Saccharomyces; adaptation; evolution; genetics; thermotolerance.

Figure 1

Barcoded RH-seq mapping of yeast thermotolerance loci. (A) Barcoded RH-seq sequencing analysis steps. Left, in a pool of S. cerevisiae × S. paradoxus hybrid hemizygotes, each harboring a transposon (gray rectangle) marked with a unique 20-mer barcode (multicolored) flanked by universal primer sites (U1 and U2), each barcode is associated with its insertion location by transposon sequencing (Tn-seq). Genomic DNA from the pool is extracted, sheared, and ligated to universal adapters (pink ovals), followed by PCR amplification with a transposon-specific primer (forward black arrow) and an adapter-specific primer (reverse black arrow) and sequencing. Right, for barcode sequencing (Bar-seq) to quantify hemizygote strain abundance after pool growth in a condition of interest, gDNA is used as input to PCR with primers to universal primer sites for sequencing. (B) Thermotolerance RH-seq screen design. An aliquot of the hemizygote pool was thawed and cultured in large format, then split into small replicate cultures, each maintained in logarithmic growth phase at the temperature of interest by back-dilution, followed by quantification by Bar-seq.

Figure 2

Hits from barcoded RH-seq mapping of yeast thermotolerance. (A) Each panel reports barcoded RH-seq results for a gene at which the S. cerevisiae allele was associated with better thermotolerance than the S. paradoxus allele, when uncovered in the hybrid background. In a given panel, the x-axis reports the log₂ of abundance, measured by RH-seq after selection at 37°C, of a clone harboring a barcoded transposon insertion in the indicated species’ allele in a given replicate, as a difference from the analogous quantity for that clone after selection at 28°C on average across replicates. The y-axis reports the proportion of observations of all clones bearing insertions in the indicated allele that exhibited the abundance ratio on the x, as a kernel density estimate. Shown are the top six genes from among all barcoded RH-seq hit loci in terms of allelic effect size; see Supplementary Table S7 for effect sizes of the complete set of hits. (B) Subcellular localization of RH-seq hit genes, where available from Pierleoni et al. (2007) and Huh et al. (2003). Genes at which effects of allelic variation on thermotolerance were reported previously (Weiss et al. 2018; Li et al. 2019) are denoted in bold type.

Figure 3

Interactions between thermotolerance loci. Each node represents a top hit gene from barcoded RH-seq mapping of thermotolerance. Each chord represents an inferred interaction, taking into account physical and genetic interactions as well as pathway membership, from the STRING database (Szklarczyk et al. 2021). Chords are weighted by the confidence of the inference of interactions; genes with higher numbers of interactions among the hits are represented by warmer colored nodes.

Figure 4

Codons under positive selection in thermotolerance loci. Each panel shows the amino acid sequence context, across type strains of Saccharomyces sensu stricto species, of codon(s) (red bar) inferred to be under positive selection along the S. cerevisiae lineage, in a hit gene from RH-seq thermotolerance mapping. Darker shading indicates a higher % identity. (A) YDR508C/GNP1, (B) YGR140W/CBF2, and (C) YMR078C/CTF18.