Deconstruction of Saccharomyces hybrids reveals multiple genomic regions required for thermotolerance

The genetic basis of phenotypic differences between species is fundamental to our view of evolution. In contrast to variation within species, phenotypic differences between species can result from a long-term sequential process whereby prior steps and multiple changes at a single locus can play important roles. However, investigating the genetic basis of phenotypes that have evolved over long time periods is limited by reproductive barriers between species. In this study, we use hybrid genetic analysis to examine reproductively isolated Saccharomyces species that have diverged in their thermal growth limits over the last 15 million years. By selectively eliminating S. cerevisiae chromosomes in hybrids with S. uvarum or S. kudriavzevii, we show that most chromosomes cause a substantial loss of thermotolerance. Reciprocal loss of chromosome 5 (Chr 5) confirms that the hybrid's thermotolerance depends on the S. cerevisiae chromosome and shows that it is partially inhibited by the S. uvarum chromosome. Using CRISPR-Cas9 to induce targeted loss of heterozygosity, we find that loss of either the left or right arm of Chr 5 results in loss of thermotolerance. Fine-mapping, complementation and reciprocal hemizygosity analysis identify MMS21, which encodes an essential subunit of the Smc5-Smc6 complex, as responsible for loss of thermotolerance associated with the left arm of Chr 5. Our findings suggest that divergence in thermotolerance over long time-periods is caused by multiple loci across the genome, with each playing a critical role in enabling growth at high temperatures.