Some of the most unique and compelling survival strategies in the natural world are fixed in isolated species1. To date, molecular insight into these ancient adaptations has been limited, as classic experimental genetics has focused on interfertile individuals in populations2. Here we use a new mapping approach, which screens mutants in a sterile interspecific hybrid, to identify eight housekeeping genes that underlie the growth advantage of Saccharomyces cerevisiae over its distant relative Saccharomyces paradoxus at high temperature. Pro-thermotolerance alleles at these mapped loci were required for the adaptive trait in S. cerevisiae and sufficient for its partial reconstruction in S. paradoxus. The emerging picture is one in which S. cerevisiae improved the heat resistance of multiple components of the fundamental growth machinery in response to selective pressure. Our study lays the groundwork for the mapping of genotype to phenotype in clades of sister species across Eukarya.
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RH-seq data have been deposited in the Sequence Read Archive (SRA) under accession SRP156210.
Orr, H. A. The genetics of species differences. Trends Ecol. Evol. 16, 343–350 (2001).
Flint, J. & Mott, R. Finding the molecular basis of quantitative traits: successes and pitfalls. Nat. Rev. Genet. 2, 437–445 (2001).
Good, B. H., McDonald, M. J., Barrick, J. E., Lenski, R. E. & Desai, M. M. The dynamics of molecular evolution over 60,000 generations. Nature 551, 45–50 (2017).
Savolainen, O., Lascoux, M. & Merila, J. Ecological genomics of local adaptation. Nat. Rev. Genet. 14, 807–820 (2013).
Nadeau, N. J. & Jiggins, C. D. A golden age for evolutionary genetics? Genomic studies of adaptation in natural populations. Trends Genet. 26, 484–492 (2010).
Wray, G. A. Genomics and the evolution of phenotypic traits. Annu. Rev. Ecol. Evol. Syst. 44, 51–72 (2013).
Masly, J. P. & Presgraves, D. C. High-resolution genome-wide dissection of the two rules of speciation in Drosophila. PLoS Biol. 5, e243 (2007).
Greig, D. A screen for recessive speciation genes expressed in the gametes of F1 hybrid yeast. PLoS Genet. 3, e21 (2007).
Eshed, Y. & Zamir, D. An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics 141, 1147–1162 (1995).
Lazzarano, S. et al. Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells. Proc. Natl Acad. Sci. USA 115, 3680–3685 (2018).
Roop, J. I., Chang, K. C. & Brem, R. B. Polygenic evolution of a sugar specialization trade-off in yeast. Nature 530, 336–339 (2016).
Steinmetz, L. M. et al. Dissecting the architecture of a quantitative trait locus in yeast. Nature 416, 326–330 (2002).
Stern, D. L. Identification of loci that cause phenotypic variation in diverse species with the reciprocal hemizygosity test. Trends Genet. 30, 547–554 (2014).
Goncalves, P., Valerio, E., Correia, C., de Almeida, J. M. & Sampaio, J. P. Evidence for divergent evolution of growth temperature preference in sympatric Saccharomyces species. PLoS ONE 6, e20739 (2011).
Salvado, Z. et al. Temperature adaptation markedly determines evolution within the genus Saccharomyces. Appl. Environ. Microbiol. 77, 2292–2302 (2011).
Sweeney, J. Y., Kuehne, H. A. & Sniegowski, P. D. Sympatric natural Saccharomyces cerevisiae and S. paradoxus populations have different thermal growth profiles. FEMS Yeast Res. 4, 521–525 (2004).
Scannell, D. R. et al. The awesome power of yeast evolutionary genetics: new genome sequences and strain resources for the Saccharomyces sensu stricto genus. Genes Genomes Genet. 1, 11–25 (2011).
Hartwell, L. H. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38, 164–198 (1974).
Mitra, R., Fain-Thornton, J. & Craig, N. L. PiggyBac can bypass DNA synthesis during cut and paste transposition. EMBO J. 27, 1097–1109 (2008).
van Opijnen, T., Lazinski, D. W. & Camilli, A. Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr. Protoc. Mol. Biol. 106, 7.16.11–7.16.24 (2014).
Parts, L. et al. Revealing the genetic structure of a trait by sequencing a population under selection. Genome Res. 21, 1131–1138 (2011).
Winzeler, E. A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).
Leuenberger, P. et al. Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability. Science 355, eaai7825 (2017).
Wilkening, S. et al. An evaluation of high-throughput approaches to QTL mapping in Saccharomyces cerevisiae. Genetics 196, 853–865 (2014).
Kim, H.S., Huh, J., Riles, L., Reyes, A. & Fay, J.C. A noncomplementation screen for quantitative trait alleles in Saccharomyces cerevisiae. G3 (Bethesda) 2, 753–760 (2012).
Sinha, H., Nicholson, B. P., Steinmetz, L. M. & McCusker, J. H. Complex genetic interactions in a quantitative trait locus. PLoS Genet. 2, e13 (2006).
Guldener, U., Heck, S., Fielder, T., Beinhauer, J. & Hegemann, J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524 (1996).
Wetmore, K. M. et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6, e00306–e00315 (2015).
Skelly, D. A. et al. Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast. Genome Res. 23, 1496–1504 (2013).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Liti, G. et al. Population genomics of domestic and wild yeasts. Nature 458, 337–341 (2009).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Nei, M. Molecular Evolutionary Genetics New York, (Columbia Univ. Press, 1987).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Loytynoja, A. & Goldman, N. webPRANK: a phylogeny-aware multiple sequence aligner with interactive alignment browser. BMC Bioinformatics 11, 579 (2010).
Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007).
The authors thank F. AlZaben, A. Flury, G. Geiselman, J. Hong, J. Kim, M. Maurer, and L. Oltrogge for technical assistance; D. Savage for his generosity with microscopy resources; and B. Blackman, S. Coradetti, A. Flamholz, V. Guacci, D. Koshland, C. Nelson, and A. Sasikumar for discussions. We also thank J. Dueber (Department of Bioengineering, University of California, Berkeley) for the piggyBac cassette. This work was supported by R01 GM120430-A1 and by Community Sequencing Project 1460 to R.B.B. at the US Department of Energy (DOE) Joint Genome Institute, a DOE Office of Science User Facility. The work conducted by the latter was supported by the Office of Science of the US DOE under Contract No. DE-AC02-05CH11231.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Fig. 1 S. cerevisiae and S. paradoxus do not differ significantly with respect to growth at 28 °C.
a, Each trace reports the mean optical density (OD595) over the time of the indicated wild isolate of S. cerevisiae (blue) or S. paradoxus (orange) cultured at 28 °C (n = 11 cultures). b, Each bar reports the mean efficiency of the indicated strain after 24 h of growth at 28 °C; individual measurements are reported as circles. Efficiencies across strains were not significantly different between the species (P = 0.07, two-sample, two-tailed t test).
Each pair of bars reports the mean number of colony-forming units per milliliter of culture per unit of cell density (OD600), from a liquid culture of S. cerevisiae DBVPG1373 or S. paradoxus Z1 grown for 24 h at the indicated temperature (n = 4 cultures). *P = 0.000152 in ANOVA with species and temperature as factors; individual replicates are reported as circles.
Each panel reports results from microscopy experiments of S. cerevisiae DBVPG1373 and S. paradoxus Z1 after 24 h of liquid growth at the indicated temperature; at 28 °C, both species were approaching stationary phase. a, Representative images. Scale bar, 5 μm. b, Each pair of bars reports the mean proportion of large-budded dyads (n = 2 cultures), for the indicated species and temperature. Individual replicates are reported as circles. See Supplementary Table 1 for exact sample numbers.
Supplementary Fig. 4 Growth of the S. cerevisiae × S. paradoxus hybrid is between that of its purebred parents at 39 °C.
Each bar reports the mean efficiency of the indicated strain (n = 4 cultures) after 24 h of growth at 39 °C; individual measurements are reported as circles. *P = 0.035, two-sample, two-tailed t test.
Supplementary Fig. 5 Dependence of the RH-seq dataset on cutoffs for read depth and transposon mutant coverage.
In each panel, the shading of a given point reports the size of the analyzed RH-seq dataset or the set of mapped gene hits, upon filtering for the indicated depth and coverage attributes using cutoff values reported on the axes. a, The x axis reports the average number of sequencing reads mapping to a given transposon insertion in either 28 °C or 39 °C selection, as a minimum level above which the insertion was retained for analysis. The y axis reports the coefficient of variation of read abundances between biological replicates for a given transposon insertion, as a maximum level below which the insertion was retained for analysis. The z axis reports the number of transposon insertions per allele, as a minimum above which the gene was retained for analysis. Shading reports the number of insertions retained for analysis in the indicated cutoff scheme. b, Data and symbols are as in a, except that shading reports the number of genes retained for analysis in the indicated cutoff scheme. c, Data and symbols are as in a, except that shading reports the number of genes that scored below a P value corresponding to a false-discovery rate (FDR) of 0.01 in the reciprocal hemizygosity test using the indicated cutoff scheme. Arrows indicate the set of cutoff values used in this study (except as noted in Supplementary Table 6), which yielded a dataset of 110,678 usable insertions across 3,416 analyzable genes, 8 of which scored below FDR = 0.01 in the reciprocal hemizygosity test.
a, Each panel reports sites in which the piggyBac transposon inserted in the indicated S. cerevisiae DBVPG1373 chromosome in clones of the S. cerevisiae DBVPG1373 × S. paradoxus Z1 hybrid, as mapped from a pool of such clones by RH-seq. Each point reports one insertion; the x axis reports the chromosomal position of a given insertion site, and the y axis reports the raw number of sequencing reads mapped to that site. Colored tickmarks along the bottom of each panel report genomic features that prohibited the mapping of reads. Read counts are from a representative RH-seq library after seven generations of culture at 39 °C, reflecting the abundance in the pool of the respective hemizygote clone harboring the insertion. b, Data are as in a, except that results are shown from transposon insertions along S. paradoxus Z1 chromosomes in the S. cerevisiae × S. paradoxus hybrid.
Supplementary Fig. 7 Variation at RH-seq hit loci has little impact on growth at 28 °C in the background of the interspecific hybrid.
Each panel reports growth efficiency measurements of targeted deletion reciprocal hemizygotes at the indicated RH-seq hit locus. In a given panel, the left-hand pair of bars reports the relative efficiencies of targeted deletion hemizygotes after culture at 39 °C, from Fig. 2b. In the right-hand pair of bars, each bar reports the mean growth efficiency (n = 12–36 cultures) after culture at 28 °C of a targeted deletion hemizygote in the indicated species’ allele, normalized by the analogous quantity for the wild-type hybrid parent; individual measurements are reported as circles. Statistical analyses of 39 °C efficiency data are reported in Fig. 2; *P ≤ 0.05, in a two-sample, two-tailed t test for a difference in efficiency between the indicated hemizygotes at 28 °C. See Supplementary Table 1 for exact P values and sample numbers.
Supplementary Fig. 8 Variation at RH-seq hit loci has little impact on growth at 28 °C in the background of the purebred species.
a, Each pair of bars reports measurements of growth efficiency of an S. cerevisiae DBVPG1373 strain harboring the S. paradoxus Z1 allele at the indicated RH-seq hit locus, relative to the analogous quantity for wild-type S. cerevisiae DBVPG1373. The dark-shaded bar reports the mean relative efficiency of the allele replacement strain after culture at 39 °C, from Fig. 3. The light-shaded bar reports the mean growth efficiency (n = 20–33 cultures) of the allele replacement strain after culture at 28 °C, relative to the analogous quantity for wild-type S. cerevisiae DBVPG1373; individual measurements are reported as circles. Statistical analyses of 39 °C efficiency data are reported in Fig. 3; *P ≤ 0.05 and **P ≤ 0.01, in a one-sample, two-tailed t test for a difference in efficiency at 28 °C between the indicated allele replacement strain and the wild-type S. cerevisiae DBVPG1373. b, Data and symbols are as in a, except that each bar reports results from an S. paradoxus Z1 strain harboring the S. cerevisiae DBVPG1373 allele at the indicated locus, relative to wild-type S. paradoxus Z1. See Supplementary Table 1 for exact P values and sample numbers.
Each panel reports a comparison of the impact on thermotolerance of allelic variation at the indicated gene, in the indicated diploid backgrounds. In a given panel, the gray bar reports the mean growth efficiency at 39 °C of a hybrid strain harboring a wild-type copy of the allele from S. paradoxus Z1 of the focal gene and a full deletion of the allele from S. cerevisiae DBVPG1373, normalized by the analogous quantity measured in a hybrid with a wild-type S. cerevisiae allele and a deletion in the S. paradoxus allele, from the insets of Fig. 2. The orange bar reports the mean growth efficiency at 39 °C of a strain of the S. cerevisiae DBVPG1373 background harboring the allele from S. paradoxus Z1 of the focal gene, normalized by the analogous quantity measured in wild-type S. cerevisiae DBVPG1373, from Fig. 3a. The blue bar reports the mean growth efficiency at 39 °C of wild-type S. paradoxus Z1, normalized by the analogous quantity measured in a strain of S. paradoxus Z1 harboring the allele from S. cerevisiae DBVPG1373 of the focal gene, from Fig. 3b. Individual measurements are reported as circles. Sample numbers for the gray, orange and blue bars are as reported in Fig. 2 insets, Fig. 3a and Fig. 3b, respectively.
Supplementary Fig. 10 At RH-seq hit loci, the effect of allelic variation is conserved across a given species and sequence divergence from S. paradoxus is a common feature of S. cerevisiae strains.
a, Each pair of bars reports the growth efficiency of an S. cerevisiae DBVPG1373 strain harboring the allele of ESP1 from the indicated strain of S. paradoxus, relative to the analogous quantity for wild-type S. cerevisiae DBVPG1373; the heights of the dark and light bars report the mean relative efficiency at 39 °C (n = 4–18 cultures) and 28 °C (n = 22–33 cultures), respectively. Individual measurements are reported as circles. b, Data and symbols are as in a, except that each bar reports results from an S. paradoxus Z1 strain harboring the allele of APC1 from the indicated strain of S. cerevisiae, relative to wild-type S. paradoxus Z1. *P ≤ 0.034 in a one-sample, one-tailed t test (39 °C) or a one-sample, two-tailed t test (28 °C) for a difference in efficiency between the indicated allele replacement strain and 1. The provenance of each strain is as follows: Z1, oak bark, UK; N17, oak exudate, Russia; IFO1804, oak bark, Japan; DBVPG1373, soil, Netherlands; DBVPG1788, soil, Finland; YPS128, soil, USA; DBVPG6044, bili wine, West Africa. c, Each row reports a comparison of the sequences of RH-seq hit loci against a genomic null. S. cer branch length is the number of sequence substitutions along the lineage leading to S. cerevisiae, in a phylogenetic tree inferred from Saccharomyces species type strains. Dxy/length is the average number of differences between the S. paradoxus type strain and a strain randomly chosen from the S. cerevisiae wine/European population, normalized by gene length. The first and second columns report the average of the indicated statistic across the eight RH-seq hit loci and across sets of eight loci randomly resampled from the genome, respectively. The third column reports the empirical P value from a test for an elevated value of the indicated statistic relative to the resampling null. See Supplementary Table 1 for exact P values and sample numbers.
Supplementary Figures 1–10, Supplementary Tables 1–4 and Supplementary Note
Abundances of transposon-mutant clones in the interspecific hybrid from RH-seq
Tests of the impact on thermotolerance of variation at each gene in turn via reciprocal hemizygote analysis of clone abundances from RH-seq
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Weiss, C.V., Roop, J.I., Hackley, R.K. et al. Genetic dissection of interspecific differences in yeast thermotolerance. Nat Genet 50, 1501–1504 (2018). https://doi.org/10.1038/s41588-018-0243-4
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