Synthetic hybrids of six yeast species

Abstract

Allopolyploidy generates diversity by increasing the number of copies and sources of chromosomes. Many of the best-known evolutionary radiations, crops, and industrial organisms are ancient or recent allopolyploids. Allopolyploidy promotes differentiation and facilitates adaptation to new environments, but the tools to test its limits are lacking. Here we develop an iterative method of Hybrid Production (iHyPr) to combine the genomes of multiple budding yeast species, generating Saccharomyces allopolyploids of at least six species. When making synthetic hybrids, chromosomal instability and cell size increase dramatically as additional copies of the genome are added. The six-species hybrids initially grow slowly, but they rapidly regain fitness and adapt, even as they retain traits from multiple species. These new synthetic yeast hybrids and the iHyPr method have potential applications for the study of polyploidy, genome stability, chromosome segregation, and bioenergy.

Fig. 1. The generation of ancestor and evolved six-species hybrids.

Synthetic hybrid generation scheme using the iHyPr method. The example shown is the six-species hybrid yHRWh39. Chromosomes were colored according to their species designation, with height representing copy number, using the sppIDer pipeline. For an extended explanation of iHyPr, including the other two crossing schemes, see Supplementary Figs. 1 and 2. Arrows mark hybridization steps. For additional intermediate and six-species hybrid nuclear and mitochondrial genomes with higher resolution, see Supplementary Figs. 3 and 4. Ancestor six-species hybrids underwent ALE for 80 generations.

Fig. 2. Genome contributions to synthetic hybrids.

The numbers and sources of chromosomes for each synthetic hybrid were inferred from sppIDer plots (Supplementary Fig. 3), which were corrected based on flow cytometry ploidy estimations. a The number of chromosomal aberrations was inferred for each synthetic hybrid as new translocation, gain, and loss events not seen in the preceding hybrid (Supplementary Fig. 3). Chromosomal aberrations involving parts of chromosomes were conservatively counted only in cases of clear fusion of entire arms, whereas smaller loss-of-heterozygosity events were not counted. The synthetic hybrids generated from each independent scheme are represented with different shapes. Color points are colored according to the number of species genomes contributing to the strain. A LOESS regression line and the 95% confidence interval of the fit are represented with a discontinuous black color and gray shadow, respectively. b Chromosome content is colored according to the species donor. Mitochondrial inheritance was inferred using mitosppIDer (Supplementary Fig. 4). The numbers of chromosomes for each species are colored according to the left heatmap legend. Incomplete and recombinant mtDNA are colored in gray. Total number of chromosomes is shown in the right part, which is colored according to the right legend. Ploidy estimates based on de novo genome assemblies, which correlates with flow cytometry (Spearman rank-sum test R = 0.88, p value = 7.5 × 10⁻⁸, Supplementary Fig. 5c), are indicated at the right side. Synthetic hybrids are reported in the order constructed (Supplementary Fig. 2). yHRW134 and yHRWh4 are shown multiple times because of their use in multiple crossing schemes. Evolved hybrids are grouped based on the conditions in which they were evolved, and they are colored according to their ancestor hybrid. Red squares highlight chromosomes that were retained or lost in all hybrids evolved in the same condition when compared to their siblings evolved in the other condition. S. cerevisiae chromosome IV, where the xylose utilization genes were inserted, is indicated by the black square. Considerable karyotypic diversity continued to be generated during 80 generations of ALE (Fig. 6), but each evolved strain is easily recognized as more similar to its ancestor six-species hybrid. Source data are provided in the Source Data file and at http://bit.ly/2v1rq1T.

Fig. 3. Characteristics of six-species hybrids.

a The number of species contributing genomes to synthetic hybrids is inversely correlated with the frequency of successful matings (n = 2 cross attempts). b Genome size is correlated with average cell area (average n = 63 counted cells). c Genome size (Supplementary Data 2) versus the average maximum growth rate (µ (n = 6 independent biological replicates), defined as (ln(OD2)−ln(OD1))/(T2–T1)) in rich medium at 20 °C (Supplementary Data 4). Dashed lines are the µ for the parent species indicated close to the line. For S. uvarum, the average of two strains with different HyPr plasmids is shown. d The maximum specific growth rate (µ, defined as (ln(OD2)−ln(OD1))/(T2−T1))) in rich medium at 20 °C is higher in interspecies hybrids inheriting S. cerevisiae mtDNA. Median values are represented by a horizontal line inside the box, and the upper and lower whiskers represent the highest and lowest values of the 1.5 × IQR (interquartile range), respectively. Colors correspond to the number of species contributing genomes to each strain. Synthetic hybrids generated from independent schemes are represented by different shapes in panels b–d. The Spearman rank-sum test R and p values are displayed. A linear regression and its 95% confidence interval are represented with a black dashed line and gray shadow, respectively. The mtDNA donor is underlined in the names in panel c. Species composition abbreviations are Scer, S. cerevisiae; Spar, S. paradoxus; Smik, S. mikatae; Sarb, S. arboricola; Skud, S. kudriavzevii; and Suva, S. uvarum. Source data are provided in the Source Data file and at http://bit.ly/2v1rq1T.

Fig. 4. Genome reduction during hybrid construction and adaptive laboratory evolution.

a The genome contribution of each Saccharomyces species is stacked, and the percentage of retention is indicated inside the bar plot for each synthetic hybrid. Presence is reported, not copy number. For example, the red stacked bar for the hybrid yHRWh24 indicates that 46% of the total unique DNA for the S. cerevisiae parent (12.07 Mbp) was detected in the hybrid, which is ~5.55 Mbp of unique DNA. Synthetic hybrids are displayed in the order constructed (Supplementary Fig. 2). yHRWh4 is shown multiple times because of its use in two crossing schemes. We did not expect 100% genome contribution for each Saccharomyces species, even for recently created hybrids, because some genomic regions (e.g. repeats) are not unambiguously detectable with Illumina sequencing data. Genome size bars are colored according to each species’ contribution. The strain names are colored based on the mtDNA inheritance inferred from mitosppIDer (Supplementary Fig. 4), with two or more mtDNAs or regions shown as a gradient. b The nuclear compositions of the S. cerevisiae parent, synthetic hybrids, and evolved hybrids are plotted according to mtDNA inheritance (n = 1 sequenced strain). Hybrids with S. cerevisiae mtDNA or with other mtDNA are colored in red and light blue, respectively. Source data are in the Source Data file and at http://bit.ly/2v1rq1T.

Fig. 5. Trait combination and improvement by adaptive laboratory evolution.

a Box plots for the individual evolved colonies isolated from YPX or YPD plates after ALE and their synthetic hybrid ancestors. Kinetic parameters were tested in 3 ml YPX on a rotating culture wheel, identically to how they were evolved for 80 generations. The average values (n = 6) of maximum specific growth rates (µ, defined as (ln(OD2)−ln(OD1))/(T2−T1)) for the S. cerevisiae reference strain (black line, yHRW135 was derived from yHRW134 by plasmid loss), ancestor six-species hybrids (purple dots), and evolved six-species hybrids (brown dots) are shown (Supplementary Data 5). Different shapes indicate the media in which the synthetic six-species hybrids were evolved. Additional kinetic parameters from microtiter plate experiments performed on evolved populations are shown in Supplementary Fig. 6 and Supplementary Data 6. Median values are represented by a horizontal line inside the box, and the upper and lower whiskers represent the highest and lowest values of the 1.5 ×  IQR (interquartile range), respectively. b Spot tests for three temperatures (22, 10, and 4 °C) are displayed for the evolved strains and the S. cerevisiae reference strain yHRW135. Evolved six-species hybrids retained the ability to grow at 4 °C, a trait not possessed by S. cerevisiae, despite the fact that it was not selected during ALE. Source data are provided at http://bit.ly/2v1rq1T.

Fig. 6. Synthetic hybrids as a tool to study genome instability.

a Box plots of the number of chromosomal aberrations inferred for ancestor and evolved synthetic hybrids (Fig. 2b, Supplementary Fig. 3) (n = 1 sequenced strain). Synthetic hybrids generated from each independent scheme are represented with different shapes. Purple and brown color points represent whether six-species hybrids were ancestor or evolved, respectively. Median values are represented by a horizontal line inside the box, and the upper and lower whiskers represent the highest and lowest values of the 1.5 × IQR (interquartile range), respectively. b For each colony isolated from the population sample of the evolved synthetic hybrid yHRWh88, the genome contribution of each Saccharomyces species is stacked, and the percentage of retention is indicated inside the bar plot. The percentage of each species’ contribution are colored according to the legend. c The number of chromosomes were inferred from sppIDer plots and corrected based on flow cytometry. The chromosome content was colored according to the species donor. The numbers of chromosomes for each species are colored according to the heatmap legend. Recombinant chromosomes are colored in gray. Asterisks indicate chromosomes that were retained in a particular colony but were not observed in the evolved yHWRh88 population sample, highlighting the instability of these hybrids. S. cerevisiae chromosome IV, where the xylose utilization genes were inserted, is indicated by the black square. Source data are provided in the Source Data file and at http://bit.ly/2v1rq1T.