Deciphering the Genic Basis of Yeast Fitness Variation by Simultaneous Forward and Reverse Genetics

Abstract

The budding yeast Saccharomyces cerevisiae is the best studied eukaryote in molecular and cell biology, but its utility for understanding the genetic basis of phenotypic variation in natural populations is limited by inefficient association mapping due to strong and complex population structure. To overcome this challenge, we generated genome sequences for 85 strains and performed a comprehensive population genomic survey of a total of 190 diverse strains. We identified considerable variation in population structure among chromosomes and identified 181 genes that are absent from the reference genome. Many of these nonreference genes are expressed and we functionally confirmed that two of these genes confer increased resistance to antifungals. Next, we simultaneously measured the growth rates of over 4,500 laboratory strains, each of which lacks a nonessential gene, and 81 natural strains across multiple environments using unique DNA barcode present in each strain. By combining the genome-wide reverse genetic information gained from the gene deletion strains with a genome-wide association analysis from the natural strains, we identified genomic regions associated with fitness variation in natural populations. To experimentally validate a subset of these associations, we used reciprocal hemizygosity tests, finding that while the combined forward and reverse genetic approaches can identify a single causal gene, the phenotypic consequences of natural genetic variation often follow a complicated pattern. The resources and approach provided outline an efficient and reliable route to association mapping in yeast and significantly enhance its value as a model for understanding the genetic mechanisms underlying phenotypic variation and evolution in natural populations.

Compensatorytrans-regulatory alleles minimizing variation inTDH3expression are common withinSaccharomyces cerevisiae

“…These strains (1) were isolated from a range of environments, (2) differ at more than 100,000 SNPs and small indels, many larger copy number variants chromosomal rearrangements, and (3) encompass much of the genetic and phenotypic diversity observed within the species (MacLean et al. ; Peter et al. ).…”

“…(D) Phylogenetic relationships among strains as estimated from genome‐wide polymorphism data (MacLean et al. ). Color of branches corresponds to estimated trans ‐regulatory effect from ancestral character estimation.…”

“…) and the most common type of polymorphism found among natural yeast strains (G→A and C→T; MacLean et al. ). We simulated the neutral evolution of trans‐ regulatory variation affecting TDH3 promoter activity by sampling these trans ‐regulatory mutational effects on TDH3 promoter activity (Fig.…”

“…; MacLean et al. ). The inserted reporter contained a copy of the 678 bp TDH3 promoter from the BY strain (entire intergenic region 5’ of the TDH3 coding sequence), a YFP coding sequence, a CYC1 terminator, and a NatMX4 drug resistance marker.…”

“…To determine variability in TDH3 expression segregating within S. cerevisiae , we used haploid, MATalpha versions of 85 natural S. cerevisiae strains created in previous work (MacLean et al. ). For each strain, we inserted a P TDH3 ‐YFP reporter at the HO locus using a standard lithium acetate transformation approach with minor alterations (Cubillos et al.…”