“Meiotic null MSH2 and SGS1 alleles in S. cerevisiae x S. uvarum hybrids result in near-haploid offspring with mixed parental chromosomal inheritance.”

“Meiotic null MSH2 and SGS1 alleles in S. cerevisiae x S. uvarum hybrids result in near-haploid offspring with mixed parental chromosomal inheritance.” | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results “Meiotic null MSH2 and SGS1 alleles in S. cerevisiae x S. uvarum hybrids result in near-haploid offspring with mixed parental chromosomal inheritance.” View ORCID Profile Taylor K. Wang , View ORCID Profile Maitreya J. Dunham doi: https://doi.org/10.1101/2025.06.02.657486 Taylor K. Wang 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Taylor K. Wang Maitreya J. Dunham 1 Department of Genome Sciences, University of Washington , Seattle, WA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maitreya J. Dunham For correspondence: maitreya{at}uw.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract In the natural world, genetic isolation between closely related species is often maintained by direct F1 hybrid sterility or hybrid breakdown in subsequent generations. This phenomenon is also observed in microorganisms within species of the Saccharomyces clade, including the model organism Saccharomyces cerevisiae . Members of Saccharomyces intermate and historical hybridization events have left genomic footprints, but these hybrids are themselves sterile and produce very few viable offspring. Previous work suggests a main cause of F1 hybrid sterility is the action of mismatch repair machinery. The sequence divergence between two parent Saccharomyces species—from ∼10% to ∼20%—is recognized by the cell as an aberrant attempt to recombine nonhomologous chromosomes during meiosis I, leading to massive nondisjunction and aneuploidy. Deletion of mismatch repair machinery improves fertility in hybrids of S. cerevisiae and its sister species S. paradoxus but leads to increased mutation accumulation during normal mitotic growth. To overcome this drawback, conditional alleles that specifically repress expression of mismatch repair machinery during meiosis have been applied in S. cerevisiae x S. paradoxus hybrids with success. Here, we describe the application of the meiotic null system to a different hybrid: S. cerevisiae x S. uvarum . The system does not increase hybrid fertility likely due to increased sequence divergence and chromosomal translocations, but mass spore recovery and flow cytometry enable the recovery of near-haploid offspring which inherit an assortment of parental chromosomes. These populations of mixed hybrid genomes can be leveraged in phenotypic studies to further understand the effects of hybrid genomes on organismal fitness. Introduction History of Saccharomyces hybrids A powerful feature of Saccharomyces cerevisiae as a model organism comes not just from the species itself, but also from its relationship to the other closely related species within the Saccharomyces clade. Although pairwise sequence divergence between S. cerevisiae and the other clade members varies up to ∼20%, S. cerevisiae is capable of cross-species mating with other clade members, resulting in interspecific hybrids. Saccharomyces hybrids exist both naturally and in domesticated settings where the pressure of natural selection is imposed by humans. There is even evidence that the entire Saccharomyces clade began with a common ancestor resulting from hybridization, followed by whole genome duplication 1 – 4 . Hybridization between two different Saccharomyces species can be an evolutionary advantage: benefits can arise from the genetic diversity and new allelic combinations that result from the combination of two parental genomes. Hybrids can also suffer from this genetic mixing, displaying incompatibilities and/or accelerated genomic changes vs. intraspecies mating due to increased instability of the hybrid genome. While Saccharomyces yeasts can form hybrids, these hybrids are typically sexually reproductive dead ends because they are largely infertile [ Figure 1A .]. Hybrid infertility is not an uncommon phenotype and can be found throughout the tree of life including in mammals, plants, and microorganisms such as budding yeasts. This infertility is viewed as one of many mechanisms that play a role in speciation. The mechanisms of hybrid infertility vary based on the taxonomy of the hybrid’s parents: in mammals Dobzhansky-Muller genomic incompatibilities commonly prevent most F1 hybrids from producing F2 offspring 5 – 8 . In contrast, the infertility of plant hybrids is usually caused by abnormal ploidy in the hybrid compared to the parents 9 , 10 . These observations were the result of decades of early study however, and the last few decades have revealed no firm rules regarding the mechanisms of speciation and hybrid infertility. Download figure Open in new tab Figure 1. Depiction of Saccharomyces hybrid sterility and underlying molecular mechanism to target strategies for increasing viable segregants. [A] Saccharomyces species readily form hybrid diploids capable of sporulating but recovery of viable offspring is extremely low. [B] Simplistic diagram comparing intraspecies Meiosis I mechanisms versus aberrant interspecies Meiosis I which results in aneuploidy and low offspring viability. [C] S. cerevisiae x S. uvarum hybrid viability results for a wild type, homozygous null allele of mismatch repair factor MSH2 , and homozygous double meiotic null MSH2 and DNA helicase SGS1 alleles. Sterility of hybrids caused by anti-recombination activity of Mismatch Repair (MMR) After determination of Saccharomyces hybrid spore inviability, the causality of this infertility became a topic of focus. There is some evidence that Dobzhansky-Muller genomic incompatibilities between parental genomes play some part in the infertility of certain hybrids, but it has been hypothesized that most hybrid infertility is due to the action of the mismatch repair (MMR) pathway during hybrid meiosis 11 . MMR machinery proteins are highly conserved across the tree of life and are critical to the cell for maintenance of genomic integrity 12 – 14 . Specifically, MMR functions to detect base - base mismatches and insertion/deletion mis-pairs generated after DNA replication and recombination. If a cell suffers double stranded DNA breaks (DSBs) during mitosis the DNA damage is repaired via homologous recombination (HR) with the help of the MMR pathway. HR functions to repair DSBs in a yeast cell by using the undamaged homologous chromosome as a template via complementary base pairing to repair the DNA damage. The proteins in the MMR pathway also work together to ensure homogeneity of template and repair strand by preventing aberrant homologous recombination. The action of MMR on sequence differences between a template and damaged chromosome during mitotic growth originates in the cell’s need to prevent sequence from an entirely different chromosome being used to repair a DSB (i.e. chr. I instead of damaged chr. IV), rather than the sequence within the homologous chromosome 15 , 16 . Improper repair of a damaged chromosome can be catastrophic for the cell due to the potential loss of critical genomic sequences, and thus MMR performs a necessary and critical function for maintaining genomic integrity during vegetative growth. MMR also plays an important role during sexual reproduction by ensuring efficacious recombination between homologous sequences between prophase I of meiosis. The quality control provided by the pathway performs a critical function in detecting proper cross overs between parental homologous chromosomes. When there is a certain level of sequence divergence between the recombining parental genomes, MMR views the parental chromosomes as homeologous instead of homologous. The amount of sequence divergence sufficient to trigger MMR recruitment is quite low: 1% divergence alone results in a statistically significant decrease in fertility for the parental hybrid. The pathway acts to abort the formation of any recombination intermediates by resolving the intermediates such that no crossover event will occur between the parental genomes [ Figure 1B .] 17 . Without recombination events among the parental genomes, there is not enough mechanical tension created by the physical connections of the chiasma between the homeologous chromosomes to properly line up on the metaphase plate before anaphase begins and chromosomes are distributed to daughter cells 15 , 18 . This mechanism is the current major hypothesis for the underlying molecular mechanism of hybrid infertility in Saccharomyces . Dysfunctional metaphase leading into anaphase causes massive aneuploidies in offspring via chromosome nondisjunction, rendering almost all inviable. The level of sequence divergence which serves to trigger MMR to block homeologous recombination is low enough that even intraspecies diploids of various S. cerevisiae strains display decreased spore viability. A sequence divergence of 2% is enough to observe a reduction of spore viability from 95% to approximately 80% 19 . The lack of sexual reproduction within Saccharomyces hybrids is a major source of frustration for the field. The lack of sexual reproduction effectively precludes hybrids and their progeny from many of the classic genetic mapping strategies used within Saccharomyces species. Without the allelic shuffling and genetic diversity among offspring that is generated during meiosis, it becomes immensely difficult to study genotype to phenotype connections in hybrids. Classical approaches like Quantitative Trait Locus (QTL) mapping, which have been applied effectively for intraspecies Saccharomyces diploids, are rendered completely useless in the context of hybrid genetics. Overcoming the obstacle of Saccharomyces hybrid infertility is the focus of a considerable body of past and ongoing work. Loss of function and conditional MMR alleles to improve hybrid spore viability A potential solution to the hybrid infertility issue was raised when mutations in the prokaryotic MMR factors mutL, mutS, and mutH were found to greatly increase the recombination rate between Escherichia coli ( E. coli ) and Streptococcus pneumonia —up to 1000X more over wild type MMR genes 20 – 23 . At the time, the homologs of MutL and MutS had been identified in S. cerevisiae as PMS1 and MSH2 respectively 24 . The functional conservation with their bacterial counterparts was suggested by several observations: both PMS1 and MSH2 mutants in yeast had an increase in the segregation frequency of genetic markers indicative of unrepaired heteroduplex DNA, similarly to the prokaryotic homologs 25 – 27 . In addition, both mutants exhibit a mutator phenotype further suggesting these strains also have poorer fidelity of DNA mismatch repair in mitotic growth as well. All of these results together prompted Hunter et. al to investigate the effect of MMR mutations and deletions of pathway members on recombination rate and hybrid fertility in Saccharomyces yeasts 18 , 24 . The group did so by mating laboratory S. cerevisiae and S. paradoxus strains and analyzing spore viability after sporulation. At the time, S. paradoxus was identified as S. cerevisiae ’s most closely related sister species, with the genomes estimated to have ∼10% divergence 26 , 28 , 29 . Of further note, karyotyping at the time had confirmed that there also existed no major structural rearrangements between the two species’ genomes, suggesting extensive synteny. These hybrid strains were then sporulated to induce meiosis via nutrient starvation and their tetrad ascospores manually dissected. The rate of surviving progeny for each strain was compared to the spore viability of the wild type hybrid, which was ∼1%. The group found that by disrupting MSH2 and PMS1 individually, spore viability for the S. cerevisiae x S. paradoxus hybrid was increased to 10.2% and 7.2% respectively. In addition to this, and further supporting the important role of anti-recombination in hybrid infertility, surviving spores were found to have at least a two-fold increase in recombination events compared to wild type. These results laid the basis for continuing research that would attempt to further increase hybrid fertility. Another more recent method that made a great stride in tackling this problem came from work by Duncan Grieg’s group 30 , 31 . Building upon the previous work in MMR factors and anti-recombination genes more generally, Bozdag et al. targeted MSH2 (the yeast MutS homolog) and SGS1 , a DNA helicase previously found to also be involved in preventing homeologous recombination. As important as msh2 mutants have been for studying the role of anti-recombination in hybrid inviability, it is critical to keep in mind that any MMR mutants exhibit a mutator phenotype. In vegetative growth these strains have an increased mutation accumulation rate. Previous work focused on reducing the mutation accumulation by limiting mitotic growth to only what was strictly necessary to gain enough cells to induce sporulation. While limiting periods of mitotic growth minimizes deleterious effects from random mutations, in an ideal world a trade off could be avoided between hybrid spore viability and mutation accumulation. One way to avoid the mutator phenotype is to turn anti-recombination genes off specifically only during meiosis so that their critical DNA repair functions during vegetative growth can be maintained. Leveraging this idea and the existence of previous meiotic null alleles, Bozdag et al. replaced the native promoters of MSH2 and SGS1 with the promoter sequence for the B-type cyclin gene CLB2 . Clb2 plays an important role during the cell cycle by promoting the transition from G2 to M phase, after which the protein is specifically and efficiently marked for degradation and its expression silenced for proper cell cycle progression 32 . By placing both SGS1 and MSH2 under the CLB2 promoter, their expression will specifically shut off only during meiosis. Without the presence of MSH2 and SGS1 , it was hypothesized that homeologous recombination in the hybrids would proceed and result in proper chiasmata formation and crossing over leading to viable progeny from the lack of chromosome nondisjunction. This is indeed what Bozdag et al. found: by creating meiotic null alleles of MSH2 and SGS1 under the control of the CLB2 promoter, S. cerevisiae x S. paradoxus hybrids spore viability increased up to 70-fold. This marked increase is almost matched to fertility rates of diverged non-hybrid crosses, and euploid offspring recovered showed multiple recombination events between each set of homeologous parental chromosomes. The drastically increased fertility and the ability to recover euploid offspring with successful genomic recombination makes it possible to perform genetic mapping in otherwise sterile hybrids. We aimed to test this meiotic null approach as a method to also enable genetic mapping in S. cerevisiae x S. uvarum sterile hybrids, which have historically been neglected as an area of study within the field of Saccharomyces hybrid fertility research. Results A msh2Δ S. cerevisiae x S. uvarum hybrid exhibits slightly increased spore viability Previous work in MMR mutants related to hybrid fertility focused on S. cerevisiae x S. paradoxus hybrids due to lower sequence divergence and full genomic synteny. This work aimed to understand how MMR mutant alleles affect hybrid spore viability in S. cerevisiae x S. uvarum hybrids with increased sequence divergence and translocations. We first tested the consequences of MSH2 deletions on S. cerevisiae x S. uvarum hybrid spore viability [ Figure 1C .]. We sporulated a hybrid diploid generated from wild type haploids from each species and manually dissected and scored progeny. After growth, a percent viability was calculated to be ∼0.87%. This value is very similar to previously reported data of ∼1% for other inter-species Saccharomyces hybrids. After repeating this experiment on the msh2Δ / msh2Δ hybrid, we observed this percent viability to increase to around 2.4%. To test spore viability for meiotic null MSH2 and SGS1 in S. cerevisiae x S. uvarum hybrids, the native promoters of MSH2 and SGS1 were replaced by each species’ CLB2 promoter. Previous comparative expression analysis was referenced to ensure conserved and specific repression of each species’ CLB2 during meiosis 33 . The viability of these offspring showed nowhere near the increase seen for the engineered p CLB2 S. cerevisiae x S. paradoxus spores: viability was much lower than for the msh2Δ hybrid strain progeny. These numbers are much smaller than the ∼10% previously observed for S. cerevisiae x S. paradoxus hybrids, which share more sequence identity and possess syntenic genomes. Reducing the action of anti-recombination increases homeologous recombination in S. cerevisiae x S. paradoxus hybrids but there exist additional barriers to hybrid fertility in S. cerevisiae x S. uvarum hybrids. Larger chromosome scale translocations have been documented between the two species’ genomes, providing an additional known barrier to hybrid fertility. However, chromosomal translocations alone cannot explain the lower hybrid fertility found in S. cerevisiae x S. uvarum hybrids so other potential factors must also contribute 31 . Meiotic null MMR S. cerevisiae x S. uvarum hybrids remain infertile but offspring aneuploidy is decreased In order to further assess the genomic composition of surviving progeny, an en masse spore retrieval protocol known as random spore analysis (RSA) was utilized. RSA is facilitated by using a parent S. cerevisiae strain from the Yeast Deletion Collection. To facilitate more high throughput experiments with this strain collection, an updated version of these strains was engineered to contain a suite of markers for use in a technique known as the Synthetic Genetic Array (SGA). These markers enable efficacious bulk selection of haploid MATa progeny from a sporulated diploid. We utilized the haploid selection features but omitted the mating type specific selection to maximize number of recovered hybrid offspring. Offspring recovery occurs by subjecting the hybrid parent sporulation culture to a combination of mechanical and chemical selections to selectively kill diploids and mechanically separate spores. A key element of the SGA marker suite for our purpose was the CAN1 gene, which makes it possible to selectively destroy diploids remaining within a sporulation culture. CAN1 encodes for an arginine permease, a transmembrane protein that facilities arginine uptake from the extracellular environment. The Can1 transmembrane protein is capable of up taking the lethal arginine analog canavanine, leading to cell death. By plating mechanically separated and chemically digested spores onto this drug, heterozygous diploids and wild type haploids are selectively killed. The presence of a single functional copy of CAN1 is capable of intaking enough toxic analogue to kill the cell, allowing both selection and counterselection of this marker. Since haploid offspring should only have one copy of either, progeny that inherit the null allele will survive on the drug because there is no permease to uptake the drug. Leveraging RSA in this way, hybrid offspring for each experimental and control strain were collected and subjected to growth assays and DNA content staining followed by flow cytometry. Analysis was performed in the FlowJo software to compare histograms of relative fluorescence units for asynchronously growing cells collected for each hybrid segregant. Specifically, these experimental segregant DNA content staining plots were compared by FlowJo fluorescence gating pairwise with the results for both a haploid and diploid control and cut offs were determined for labeling as near-haploid, aneuploid slight, or aneuploid severe [ Figure 2A , Figure 2B .]. The segregants were then binned into several different categories based on the overlap, or lack thereof, of the experimental samples with the haploid control [ Table 1 ]. These flow cytometry results were used to calculate a percentage of near-haploid offspring for each hybrid population: wild type, and two different biological replicates of the homozygous p CLB2-MSH2 and p CLB2-SGS1 engineered strain [ Figure 2C .]. As expected, the wild type hybrid strain had the most aneuploid offspring, with the near-haploid percent at only 7.89%. The biological replicates for the experimental p CLB2 promoter replacement hybrids yielded a substantially larger percentage of near-haploid offspring at 46.5% and 43.9% respectively. In addition, the aneuploid offspring produced by these p CLB2 replacement strains were on average less aneuploid, indicated by the progeny classified as slight aneuploid vs severe. View this table: View inline View popup Download powerpoint Table 1. Flow cytometry ploidy binning summary for recovered progeny of wild type and MSH2 SGS1 meiotic null S. cerevisiae x S. uvarum hybrids. Download figure Open in new tab Figure 2. Comparative DNA staining flow cytometry analysis of hybrid offspring ploidy between wild type and p CLB2 MMR meiotic null strains. [A] Representative plots are shown for binning of p CLB2 progeny as Severe or Slightly aneuploid. [B] Offset overlay showing in more detail the average difference in aneuploidy levels for p CLB2 offspring vs. wild type offspring. [C] Comparison of spore viability for diploid hybrid strains. All strains are homozygous at all MMR alleles. Total number of offspring analyzed: 38 MSH2 SGS1 , 43 p CLB2 -MSH2 p CLB2 -SGS1 clone 1, 41 p CLB2 -MSH2 p CLB2 -SGS1 clone 2. Whole genome sequencing of select progeny reveal patterns of chromosome inheritance Based on the results of the flow cytometry DNA content screens, we selected 16 p CLB2 -MSH2 p CLB2 -SGS1 hybrid strain progeny that appeared near-haploid for 10X low coverage whole genome sequencing (WGS). The low coverage sequencing was sufficient to properly map reads and determine relative copy number between the two parental genomes, given the 20% sequence divergence between them. Analyzing the alignment of these reads to a hybrid parent genome revealed that almost all the progeny had inherited a complete set of 16 chromosomes with limited aneuploidies. Interestingly, we did not see uniparental inheritance: instead each progeny inherited each chromosome mostly in its entirety from either the S. cerevisiae or S. uvarum parent or both in the cases of aneuploidies [ Figure 3A .]. Recombination between the hybrid genomes was rare, observed in only a single recovered near-haploid offspring from a meiotic null parent. Particular attention was directed to the inheritance patterns for chromosomes that contain one of the translocations present between the S. cerevisiae and S. uvarum genomes: reciprocal for Chr II and Chr IV, Chr VIII and Chr XV, Chr VI and Chr X, and nonreciprocal for left arm of Chr VII onto Chr V. The presence of homeologous sequence tracts between these translocated parental chromosomes resulted in ubiquitous coinheritance for each pair across the 16 near-haploid progeny reported here. Either the hybrid progeny inherited both of one chromosome and a single chromosome from one parent, or it inherited parent matched chromosome pairs: for example both Chr II and Chr IV from S. cerevisiae only [ Figure 3B .]. Based on previous studies in fungi for the consequences of translocations during meiosis, this finding aligns with the expectation that during meiosis chromosomes with homeologous translocated sequences will form quadrivalents on the metaphase plate and thus cosegregate during anaphase I 34 . Download figure Open in new tab Fig. 3. Summary view of parental chromosome inheritance for viable S. cerevisiae x S. uvarum offspring. [A] Copy number variation plots from alignment of WGS reads to concatenated S. cerevisiae and S. uvarum hybrid genome. Representative offspring from wild type and either engineered clonal isolate showing patterns of coinheritance and a recombination event. [B] Summary plot of parental chromosome inheritance for the 16 viable meiotic null offspring recovered. Other striking patterns observed across the 16 near-haploid progeny (Supplementary file 1) were observed for parental chromosome inheritance ( Figure 3 ). One example is that all progeny recovered had only one copy of Chr V, and it was always inherited from the S. cerevisiae parental genome. This pattern is expected due to the use of canavanine to select for the can1 allele from S. cerevisiae , as described above. This provides a method to bias parental chromosome inheritance from meiotic null S. cerevisiae x S. uvarum parents. Usage of selectable and counterselectable markers could also be used to select for single chromosome inheritance for chromosomes that show enriched aneuploidy and double inheritance like Chr X. Selecting for uniparental inheritance of these chromosomes could be used in future studies to inform the mechanism of coinheritance of certain aneuploidies. Another example of strong parental bias for chromosome inheritance was observed for Chr XII, where over 85% of viable S. cerevisiae x S. uvarum offspring had inherited only the S. uvarum chromosome XII, and no progeny inherited both parental Chr XII. This finding is particularly intriguing due to the presence of the multicopy rDNA array on the right arm of Chr XII for both S. cerevisiae and S. uvarum . The rDNA exists as a tandem array of multiple copies, with an observed range of approximately 100-200 copies in most S. cerevisiae isolates. Each single copy encodes for the RNA transcript components of the cell’s ribosomes: the nucleoprotein complex responsible for translation of the proteome from RNA transcripts. Previously, work on understanding the phenotypic consequences of rD variation and its role in the cell’s biological functions had focused almost exclusively on building this understanding within intraspecies variation among S. cerevisiae isolates with little consideration of rDNA phenotypic effects in Saccharomyces hybrids. While the viability of the parent S. cerevisiae x S. uvarum hybrid suggests no fundamental incompatibility between S. cerevisiae or S. uvarum rDNA and the rest of the hybrid genome, it is possible that the process of undergoing meiosis provides a selection pressure which reveals an incompatibility resulting in biased inheritance of the S. uvarum Chr XII in viable hybrid progeny. The strong signature of S. uvarum Chr XII inheritance bias, even for a small population, is striking and points at potential biology significance. Assessing aneuploidy of parental chromosome bias across the collection of near-haploid genomes reveals suggested inheritance patterns as well. We observed an enrichment (almost a third of recovered progeny) in biparental aneuploidy for chromosomes VIII and IX. For uniparental inheritance bias (defined here as greater than 2:1 ratio of parental inheritance per chromosome with minimal aneuploidy), we observed a bias for S. cerevisiae parental inheritance for chromosomes II, IV, V, XIII, and XVI. An enrichment for S. uvarum parental inheritance was observed for chromosomes XII—the chromosome on which the rDNA locus resides—XIV, and XV. Other chromosomes were observed to be inherited equally from either parental genome, including chromosomes I, III, V, VII, X, and XI. We do not currently understand the reasons for these patterns or yet have an understanding of the instances of genetic conflict that could potentially be occurring. These represent research areas of interest to be further pursued with this engineered hybrid system. Discussion The work presented here examined the effect of mutant alleles for mismatch repair factors on hybrid spore viability for interspecies S. cerevisiae x S. uvarum hybrids. Previous work has focused solely on assessing the spore viability effect of suppressing the anti-recombination activity of MMR in hybrids of S. cerevisiae and its sister species S. paradoxus . Meiotic null alleles of the MMR factor MSH2 and DNA helicase SGS1 were previously observed to increase spore viability of S. cerevisiae x S. paradoxus from less than 1% to approximately 35% with multiple recombination events in the hybrid genome during meiosis. Here we report that replicating the meiotic null alleles in S. cerevisiae x S. uvarum hybrids revealed no increase in fertility. While disappointing for efforts to genetically map traits for hybrids, the stark difference in spore viability for S. cerevisiae x S. paradoxus versus S. cerevisiae x S. uvarum hybrids is not entirely surprising. In S. cerevisiae x S. paradoxus hybrids, the parental genomes have less sequence divergence at ∼10% (∼20% for S. cerevisiae vs S. uvarum ) and are more syntenic (3 reciprocal and 1 nonreciprocal translocations for S. cerevisiae vs S. uvarum ) 35 . Previous studies that have examined the effect of translocations on hybrid sterility and more recent work in intra-species matings of lab and natural S. cerevisiae isolates have shown that translocations have a separate and significant impact on hybrid fertility from genomic sequence divergence. Further supporting this hypothesis is a study where the authors found that in a screen of intra-species S. cerevisiae crosses between the lab S288C reference strain and natural isolates the subsequent F1 hybrids with the lowest fertility all contained translocations in reference to the S. cerevisiae genome 36 . This intra-species crossing screen also revealed that progeny viability was not correlated with sequence divergence for these intra-species hybrids, but instead found reproductive isolation was mainly related to genomic rearrangements. In this scenario, the effect of sequence divergence between parental genomes is minimized and it becomes clear that in the case of low sequence divergence genomes, translocations alone do contribute to driving hybrid infertility. We believe that a similar effect is at play in S. cerevisiae x S. uvarum hybrid fertility, and could potentially suggest an explanation for the lack of offspring even after the p CLB2 MMR meiotic null alleles: by alleviating the action of mismatch repair as a cause of hybrid infertility, the impact of multiple reciprocal and nonreciprocal translocations remain and still severely impact hybrid meiosis in a hybrid with greater sequence divergence (20%) then previously examined for the effect of translocations between hybrid genomes on spore viability. These previous results and the work reported here suggest that the effects of translocations and sequence divergence between two Saccharomyces genomes can be overlapping and independent in their severity of effect for hybrid spore viability. The lack of any recovered viable progeny from manual dissections of p CLB2-MSH2 p CLB2-SGS1 S. cerevisiae x S. uvarum hybrids is striking. The molecular mechanism for this lack of progeny when compared to MSH2 SGS1 hybrids is unknown. It could be speculated that due to the critical action of Sgs1 in resolving meiotic recombination intermediates to non crossovers for non homologous sequences, the loss of this function during S. cerevisiae x S. uvarum hybrids has particularly detrimental consequences due to the additional problematic nature of translocations and increased sequence divergence between the parent genomes when compared to the p CLB2 -SGS1 allele in S. cerevisiae x S. paradoxus hybrids. In future pursuits it would be informative to dissect patterns of viability and percent near-haploid offspring for both p CLB2 -MSH2 and p CLB2 -SGS1 single meiotic null hybrid strains. These single meiotic null hybrids will enable interrogation of the underlying molecular mechanisms for the total lack of recovered viable offspring during manual dissection by separating potential effects of meiotic null SGS1 from MSH2 . Understanding the deeper reasons for lack of fertility increase in these engineered S. cerevisiae x S. uvarum hybrids could also potentially lead to other approaches that will provide an increase in fertility. While the engineered promoter replacement S. cerevisiae x S. uvarum hybrids may not have yielded an increased percentage of viable spores by manual dissection, the use of higher throughput random spore analysis and flow cytometry shows that spores recovered from the engineered strain show a significant increase in near-haploid offspring compared to the progeny generated from the MSH2 SGS1 wild type hybrid strain. It should be noted that the particular random spore method applied here yielded a significant proportion of diploid parental strain yeast which survived the vegetative cell killing treatment. While a more stringent spore selective protocol can be utilized to overcome this drawback, it is also possible to sort away this population by flow cytometry while selecting for near-haploid offspring. A serious limitation of this study was the number of near-haploid progeny studied. While none of the progeny recovered were perfectly haploid, there exists the possibility that with a higher number of examined offspring truly haploid offspring with mixed chromosomal inheritance could be recovered and subjected to further study. With the meiotic null S. cerevisiae x S. uvarum hybrids it is also possible to bias parental chromosome inheritance and select against biparental inheritance, as evidenced by the S. cerevisiae Chr V unilateral inheritance due to the usage of the CAN1 gene as a selectable and counter selectable marker during the mass spore recovery protocol. Similarly, use of the SGA mating type-specific selection markers could bias Chr III inheritance, and also generate populations consisting of a single mating type, facilitating pooled experiments. Meiotic null engineered hybrids in tandem with mass plating methods and sorting flow cytometry empower recovery of a population of viable near-haploid progeny that have mixed inheritance of S. cerevisiae and S. uvarum chromosomes [ Figure 4 .]. Individual isolates or the pooled library of mixed chromosome near-haploid offspring can then be screened or phenotyped to study trans acting genetic background effects and the genomic consequences of mixed parental chromosomal inheritance in hybrid offspring. In conclusion, we present here a method by which MMR meiotic null S. cerevisiae x S. uvarum hybrids can be sporulated and their surviving near-haploid progeny can be isolated through mass plating methods and flow cytometry to enable the application of genetic mapping methods to infertile Saccharomyces hybrids separated by genomic translocations. Download figure Open in new tab Figure 4. Diagram depicting workflow with constructed meiotic null S. cerevisiae x S. uvarum hybrid strain undergoing sporulation, recovery of surviving offspring with mass plating method, and flow cytometry sorting on DNA content to isolate near-haploid collection of progeny for further screening and phenotyping. Materials and methods Strains Detailed strain genotype information found in Supplemental table 1. For the msh2 knockout strains, the S. cerevisiae haploid was sourced from the MATa Yeast Deletion Collection. The creation of the SGA version of this collection was described previously in Tong et al., but in short, each non-essential gene was systematically deleted by a barcoded KanMX cassette within a diploid strain derived from BY4742 already containing an assortment of other markers. Subsequent sporulation of this diploid and selective screening of progeny allowed for recovery of haploid offspring with the SGA marker suite, and the gene of interest deletion cassette. The msh2Δ knock out for S. uvarum was generated previously by lab member Monica Sanchez. Briefly, the endogenous genomic locus of MSH2 was replaced by transforming a wild type S. uvarum yeast strain derived from CBS7001 with a PCR product containing the NatMX selectable marker flanked by primer designed homology to the endogenous MSH2 locus. S. cerevisiae x S. uvarum hybrids were generated by patch mating of parental strains on solid YPD plates and picking zygotes under the microscope after 4-5 hours post mating. ‘wild type’ hybrid was generated by mating a haploid version of the SGA strains termed YMD895 to the haploid S. uvarum strain termed YTW2. The haploid MSH2 and SGS1 S. cerevisiae strains used in this work were generated using a diploid S. cerevisiae parental strain originating from the synthetic genetic array (SGA) collection originally generated by Tong et al. This strain had the genotype of n Δ:: TE -Sp_his5 / CAN1 ly Δ / LYP1 h 3Δ / h 3Δ l Δ0 / l Δ0 3Δ0/ 3Δ0 t5Δ0/ LYS2/LYS2 ET 5/met 5Δ and was made from BY4742, which is isogenic to the reference laboratory S. cerevisiae strain S288C. A diploid strain termed YTW3 was used to avoid any suppressor mutations in case of fitness defect in the promoter replacement allele that could occur in a haploid. Each promoter replacement strain was made by transforming the yeast with the appropriate PCR product (plasmid assembly defined below) as described previously 37 . Briefly, a colony of the parent S. cerevisiae strain YTW3 was grown overnight. This culture was diluted the next morning and grown until an OD of 0.5 – 0.8 was reached. Cells were then spun down, washed, and subjected to a chemical transformation using lithium acetate, polyethylene glycol, water, and DNA to be transformed. Cells were then recovered for two hours in YPD and finally plated on either D + G418, or D + CloNAT plates as needed. Single colonies were picked to check for promoter replacement via PCR and Sanger sequencing. S. uvarum strains in this study all originated from the CBS7001 parental strain, whose HO locus has been disrupted to prevent mating type switching. Promoter replacement strains were made similarly to the S. cerevisiae strain: transformation of the diploid termed YTW8 with PCR to replace either the MSH2 or SGS1 promoter with the S. uvarum CLB2 promoter marked with a NatMX and KanMX cassette respectively. The only change in protocol was for the heat shock to occur at 37°C instead of 42°C, and for all normal growth to occur at 25°C instead of 30°C due to the difference in preferred growth temperature for S. uvarum compared to S. cerevisiae . Gibson assembly To replace the native promoters of SGS1 and MSH2 , primers were designed to amplify the CLB2 promoter sequence from genomic DNA of both S. cerevisiae and S. uvarum . These PCR products were then cloned into the plasmid pFA6a via Gibson assembly using inverse PCR for the plasmid backbone to remove several expression cassettes unnecessary for this experiment and designed overhangs. or each species’ promoter, two constructs were created: one with a KanMX cassette upstream of the promoter, and the other with a NatMX cassette. The KanMX cassette was used to demarcate the replacement of the native SGS1 promoter with p CLB2 , and the NatMX marker used to indicate promoter replacement for MSH2 loci in both species. Spore viability Hybrid cultures were sporulated in 5 mLs of 1% potassium acetate, 0.05% dextrose, and 0.1% yeast extract for 4-7 days. scospore dissection was carried out by spinning down 5 μL of the sporulation culture, resuspending in 5 μL of yeast lytic enzyme YLE at 100 U/mL in 1 M sorbitol, and plating on YPD after a 30 minute digestion at 30°C. Dissections were carried out at room temperature on a Nikon H550S Eclipse 50i scope via manual needle dissection. After 3 days of growth at 25°C surviving tetrad spores were counted. Percent viability for each hybrid strain was calculated by the following formula: . To recover viable progeny en masse in a high throughput setting, the method of random spore analysis was used 38 . Briefly, a volume of sporulation culture for each hybrid was spun down and resuspended in μL of /mL YLE. After 30 minutes of incubation at 30°C, cultures were subjected to 1 minute of vortexing followed by plating onto C +Canavanine -arginine -serine. Surviving colonies were collected, preserved at -80°C in glycerol stocks, and subsequently subjected to growth analyses and flow cytometry Flow cytometry DNA content flow cytometry was performed using a BD Accuri C6 flow cytometer. The protocol is as described previously in the 2015 edition of Methods in Yeast Genetics and Genomics (A Cold Spring Harbor Laboratory Course Manual). Briefly, spores as well as haploid and diploid controls were seeded to grow overnight at the appropriate temperatures. Cultures were back diluted to ∼0.2 OD the next day and then harvested between ∼ 0.5 – 0.8 OD by spinning down 1 mL and resuspending in 1 mL of 70% ethanol. Half of these fixed cells were then washed with water and subjected to RNase A digestion in μL of 5 m Tris-HCl (pH = 8.0) for 2 hours at 37°C. Samples were then spun down and resuspended with mg/mL roteinase K in μL of 5 m Tris-HCl (pH = 7.5) for 30 minutes at 5 °. fter another spin, samples were resuspended in μL of S buffer (200 mM Tris-HCl pH = 7.5, 200 mM NaCl, and 78 mM MgCl 2 ). Directly before samples were loaded onto the cytometer, μL of cells were transferred to a 96-well plate containing μL of Y green diluted 5 X in 50 mM Tris-HCl (pH = 7.5) and subjected to sonication. All samples were sonicated for 3 seconds at 10% power, resulting in a 5-10 Watt power output. Analysis of flow cytometry samples, including gating for debris and doublets, was carried out in FlowJo. WGS Sequencing Preparation and Analysis The sequencing libraries for the near-haploid hybrid offspring were prepared using a commercial Illumina NextSeq library sequencing kit. The quality and size of the libraries was verified by performing gel electrophoresis on a 5% SDS-PAGE gel for 40 minutes at 150 V followed by staining and imaging. An average 10X sequencing depth was achieved across each S. cerevisiae x S. uvarum near-haploid offspring genome. Final reads were processed as follows: Trimmomatic was used to remove any remaining adapter sequences, alignment of trimmed reads to a concatenated S. cerevisiae x S. uvarum genome was carried out by the Burroughs-Wheeler Aligner package (BWA-MEM), sorted using PicardTools, multimapping reads removed by a custom script, and the samtools package was then used to generate read depth files for each strain. Final copy number plots for all hybrid progeny can be found in Supplemental file 1. Sequencing Analysis Pipeline for Copy Number Variation Read depth files from the initial analysis detailed above were then used as input to a custom copy number variation script written in RStudio. In brief, alignment files were imported to RStudio as data frames while simultaneously removing mitochondrial DNA and read depth at each base pair position was normalized to average genome wide read depth. 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