Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion
[['Genetic diversity relies on diversification of the parental genome information .', 'Besides spontaneous and environmentally induced de novo mutations , sexual reproduction is the prominent source of genetic diversity: it reshuffles the genetic information among individuals from a given species , creating the new combinations of alleles upon which the Darwinian selection will potentially act .', 'Thus , the genetic diversity of a given population depends on the random mating of the gametes , the capacity of meiosis to promote homologous recombination between the polymorphic parental chromosomes as well as to ensure the random segregation of the chromosomes in the gametes .', 'The meiotic developmental program involves the segregation of the homologous pairs of sister chromatids to opposite poles at the first meiotic division ( Meiosis-I or reductional division ) , followed by the segregation of the sister chromatids at the second meiotic division ( Meiosis-II or equational division ) , which is followed by the differentiation of gametes , or spores in yeast ( Fig 1 ) [1] .', 'Another hallmark of meiosis is the high level of inter-homologs recombination during the prophase-I of meiosis .', 'Meiotic recombination is not evenly distributed along the chromosomes but inter-homolog recombination occurs at least once per chromosome [2] .', 'This is initiated by the formation of programmed Spo11-dependent DNA double-strand breaks ( DSBs ) .', 'Afterwards , inter-homolog repair of these DSBs results in the formation of crossovers ( CO ) and non-crossover ( NCO ) recombinant products [3] .', 'The relative outcome of CO and NCO events is genetically controlled , depending on the processing of the recombination intermediates and multiple regulatory pathways [4] .', 'Importantly , the crossovers that physically link each pair of homologs ensure the proper reductional segregation at Meiosis-I [5] which ultimately leads to the halving of the genome content and the formation of viable haploid gametes , or spores .', 'Defects in meiotic recombination can arrest the progression of meiosis and are a source of genomic abnormalities and therefore sterility .', 'Notably , the frequent spontaneous formation of disomic chromosome 21 gametes in the male or female gametogenesis is the cause of Down syndrome in humans [6] .', 'In sharp contrast , in all eukaryotes , recombination between the homologous chromosomes is rare in somatic cells [7 , 8] .', 'Accidental DSBs are preferentially repaired by Non-Homologous End-Joining , a mutagenic process , or repaired in the G2 phase of the cell cycle by homologous recombination between the identical sister chromatids , being promoted by the existence of sister chromatid cohesion that favors recombination between the sisters at the expense of homologs [9 , 10] .', 'Thus , the rarity of inter-homolog mitotic recombination contributes to the clonal perpetuation of the parental allelic combinations .', 'Here , to isolate diploid recombinants in yeast , we used the singular , yet remarkable property of Saccharomyces cerevisiae diploid cells to exit from the prophase-I of meiosis and be able to re-enter into mitosis , a puzzling process termed “Return to Growth ( RTG ) [11–14] .', 'As illustrated in Fig 1 , budding yeast diploid MATa/MATα cells are induced to enter meiosis by nutritional starvation [1]; then , the cells enter into S phase and the chromosomes replicate .', 'Next , ~160–200 Spo11-dependent DSBs occur per cell [15] and are efficiently repaired by homologous recombination before the MI reductional division occurs .', 'Remarkably , the highly differentiated and coordinated progression of the DNA intermediates and changes in chromosomal structures through the prophase-I of meiosis is reversible by the addition of rich medium , up to the irreversible commitment point that precedes the reductional division step , thus after the time of DSB formation .', 'This remarkable transition from the meiotic prophase-I to mitosis is under the regulatory control of the Swe1 kinase , which modulates the Cdk1 activity [16] .', 'This kinase permits an unusual progression of the mitotic cell-cycle events , allowing the induction of bud formation in the absence of re-replication [16 , 17] .', 'Thus , upon RTG , the diploid cell ( hereafter called the mother cell ) that entered meiosis and experienced DSB formation with or without repair will exit with or without a recombined 4C genome that will segregate equationally , leading to the random segregation of two non-sister chromatids in the mother cell while the other two chromatids will segregate in the daughter cell .', 'Using a single locus intragenic heteroallelic assay , several authors [11 , 13] observed that the RTG cells were much more frequently recombined than vegetative cells , strongly supporting the conclusion that these recombination events were initiated in meiosis .', 'More recently , physical analyses of the HIS4-LEU2 hotspot showed that in wild type cells the meiotic Spo11-DSBs are rapidly repaired upon RTG ( within 2 h ) [17 , 18] and that the Joint Molecule intermediates ( JMs ) that accumulate in the prophase-arrested ndt80Δ mutant are well repaired upon RTG , but in contrast to meiosis , preferentially give rise to NCO recombinant molecules rather than CO recombinant molecules [17] .', 'Mutant analyses showed that RTG recombination was dependent on the Rad51 strand exchange protein but not Dmc1 [18] and that most JMs are repaired by the Sgs1 pathway that produces only NCOs , while a fraction of JMs are repaired by the Mus81/Mms4 pathway producing both NCOs and COs[17] .', 'But so far however , very little is known about the outcome of the RTG process on the architecture of yeast hybrid genomes .', 'Here , we report the whole genome sequencing of 36 RTG strains derived from the S288c/SK1 S . cerevisiae hybrid .', 'We found that the RTG strains are bona fide diploids , diversely recombined both in terms of frequency and location .', 'Furthermore , as a proof of principle , we performed a genotype/phenotype analysis of the RTG strains for three Mendelian and one complex traits .', 'This validates the potential of this previously unappreciated procedure of genome diversification to rapidly map quantitative traits loci ( QTLs ) in diploid strains , without the necessity to undergo sexual reproduction .'], ['To examine the genome-wide recombination profile of RTG cells , we constructed a yeast diploid hybrid ( AND1702 ) by mating two S . cerevisiae haploid strains from different genetic backgrounds , S288c and SK1 ( S1 Table ) .', 'A similar but differently marked S288c/SK1 hybrid was previously used for meiotic tetrad analyses [19] .', 'Overall , the diploid contains >62 , 000 single nucleotide polymorphisms ( SNPs ) , distributed along the 16 homologous chromosomes ( S1A Fig ) resulting in a genome wide divergence of ~0 . 7% [20 , 21] .', 'The average inter-SNP distance is 191bp ( S1B Fig ) .', 'Few long regions are devoid of polymorphic SNPs ( 11 regions ≥10kb ) .', 'Therefore , this hybrid strain is ideal to achieve high-resolution genotyping and therefore to map recombination events .', 'The strain also carries several auxotrophic markers , appropriate for screening the RTG cells ( see below ) .', 'The S288c/SK1 hybrid strain sporulates efficiently ( 88% of asci after 48 h in the sporulation medium ) , like the SK1 strain and more than the S288c diploid ( S2A Fig ) .', 'However , it produces tetrads with reduced spore viability ( 71% ) relative to both diploid parents ( S2B Fig ) .', 'The distribution of viable spores per tetrad is reported in S2C Fig . The hybrid produces 4 viable spore tetrads ( 43% ) but also a significant fraction of 3 , 2 , 1 and 0 viable-spores tetrads are observed .', 'Several factors may reduce spore viability [22] .', 'Most likely , an incompatibility between the S288c and SK1 alleles may impair germination and/or growth capacity .', 'In some instances , residual growth observed as micro-colonies are seen under the microscope .', 'An alternative , non-exclusive , hypothesis is the occurrence of Meiosis-I or Meiosis-II chromosome mis-segregations , leading to unviable spores with aneuploid genomes .', 'In order to isolate the RTG cells , we used two complementary methods illustrated in Fig 1 . The first method , “isolation by prototroph selection” , corresponds to the traditional RTG plating assay [11 , 13] based on the selection of intragenic recombinants , in this case arginine prototrophs ( Materials and Methods ) .', 'The limitation of this selective approach is that upon RTG recombination , only one of the arg4 alleles is converted to ARG4 and therefore only the mother or the bud ( daughter cell ) that inherits the wild type recombinant allele is recovered after RTG .', 'To overcome this limitation , we devised an alternative single cell micromanipulation method to isolate mother cells , that derived from meiosis upon return to growth , from their first daughter cell that arises upon bud formation ( Fig 1 , Materials and Methods ) .', 'This micromanipulation method offers two advantages over the prototroph selection .', 'First , it eliminates cells committed to complete meiosis , as they do not form a bud upon transfer to rich medium ( they ultimately form tetrads ) .', 'Second , since re-replication does not occur before budding [16 , 17] , all four chromatids present in the “returned” meiotic cell are recovered in the pair of mother-daughter cells , similar to the recovery of the four products of meiosis in a tetrad .', 'Thus , in RTG pairs , any anomalies in chromosome segregation and marker segregation , including the gene conversion events , can be identified as in four-spores tetrad analyses .', 'To isolate recombinant RTG cells , the meiotically induced diploid cells should be retrieved at prophase-I of meiosis , after DSB formation and before their commitment to complete meiosis .', 'To determine this time window in the hybrid background , relatively to the SK1 and S288c backgrounds , we monitored and compared several landmark parameters of the meiotic progression in time course experiments .', 'First , we monitored the progression of the cell population during sporulation by DAPI staining of the cell nuclei in the three backgrounds .', 'We observed that the kinetics of meiotic progression of the hybrid resembles more the one of SK1 than of S288c , and that in the hybrid , ~50% completed the Meiosis-I divisions at t = 8 h ( S3A Fig ) .', 'Second , we performed a physical analysis of meiotic DSBs and recombination products formation at the ARG4/DED81 hotspot of recombination located on chromosome VIII ( S3B–S3D Fig ) .', 'DSB formation at the DSB1 and DSB2 sites [23] is first detected at t = 3 h , similar to SK1 ( S3C and S3D Fig ) .', 'Next , the meiotic recombinant products ( R1 and R2 ) are first observed at t = 5 h and accumulate until 8 h ( S3C and S3D Fig ) .', 'We conclude that , in the cell population , the initiation and completion of meiotic recombination in the hybrid background occur between 4 and 8 hours .', 'Third , we directly examined the occurrence of recombinants in the hybrid by performing a time course RTG experiment .', 'After induction of sporulation for various times in liquid medium , we plated the RTG cells on–Arg plates to select arginine prototroph colonies resulting from intragenic recombination between the arg4-RV and arg4-Bgl heteroalleles [24] .', 'The production of arginine prototrophs arise between t = 4–8 h ( S3E Fig ) , that are enhanced by at least three orders of magnitude , as compared to the basal mitotic level of Arg+ colonies observed at t = 0', 'h . Finally , in the hybrid , since crossovers formed between the heterozygous recessive alleles at HIS4-LEU2 , HIS3 and MET15 loci and their respective centromeres induce loss of heterozygosity ( LOH ) in RTG ( see below ) , we examined the appearance of auxotrophic colonies in the RTG time course ( Materials and Methods ) .', 'After induction of sporulation for various times , we plated the RTG cells on rich glucose medium and then replica plated the colonies on media lacking histidine , leucine or methionine .', 'Auxotrophic colonies start to appear after 4 h of incubation in the sporulation media and their frequency greatly increases up to 8 h ( S3F Fig ) .', 'Based on these data , the 4–8 hour time window was used for the RTG experiments .', 'A potential drawback in analyzing a small number of cells from the meiotic time course could be that the cells are not at the expected meiotic stage , due to the relative asynchrony of the meiotic progression .', 'Here , within the 4-8h time window , we observed that an increasing fraction of cells isolated by micromanipulation progressed to give a tetrad ( S3G Fig ) , indicating that those cells had passed the commitment point to irreversibly complete meiosis and sporulation , in a proportion consistent with the kinetics of meiotic progression ( S3A Fig ) .', 'Also , as expected [11–14] [16 , 17] , the vast majority ( >94% ) of cells that budded gave rise to two viable cells , forming mother and daughter colonies .', 'In the remaining cases , only the mother or daughter cell was viable .', 'Several hypothesis can explain this asymmetric lethality; For example , a technical consequence of separating the mother and daughter cells upon micromanipulation , genetic incompatibilities resulting from loss ( es ) of heterozygosity in one of the cell or any defect in the process of RTG , independent of the budding process .', 'The rarity of these cases prevented us to further analyze them .', 'In addition , we observed that a significant number of the isolated cells ( 61% ± 18% , mean ± SD ) did not bud nor sporulate ( S3G Fig ) .', 'To eliminate the hypothesis that this lethality is due to the RTG process per se , we examined the viability of unbudded cells isolated at earlier time points of meiosis as well as the viability of the vegetative hybrid and SK1 parent cells grown in rich YPD medium and in the pre-sporulation SPS medium .', 'Again , in all cases , a similar proportion of unbudded cells placed on YPD medium by micromanipulation did not grow , indicating that this cell lethality is not meiosis- nor strain-specific ( S3G Fig ) .', 'Other studies have also reported this observation that , after micromanipulation , a high proportion of cells do not divide , especially when cells are isolated from non-logarithmic vegetative culture [25] or from meiotic cultures [26] , compared to when cells were isolated from logarithmic vegetative cultures .', 'We do not know the cause of this cell lethality , but in all conditions , the cells that remained on the inoculum area seemed to undergo normal mitotic divisions , suggesting an effect of the micromanipulation .', 'Altogether , we conclude that the meiotic cells that bud after RTG are in most instances viable and as shown below , properly segregate their chromosomes , giving rise to viable euploid cells .', 'We analyzed 36 RTG strains subjected to high throughput whole genome sequencing ( Materials and Methods ) .', 'Six strains ( RTG1-S to RTG6-S ) were isolated by Arg+ selection ( method 1 ) and 30 strains ( RTG7-M/-D to RTG21-M/-D ) were isolated by mother-daughter dissection ( method 2 ) .', 'Their phenotypes were determined with respect to mating and growth on the Arg , His , Leu and Met depleted media and confirmed by the sequencing data .', 'The genetic marker genotypes are shown in S2 Table .', 'Next , the genotype at SNP positions and recombination profiles were extracted using a dedicated bioinformatic pipeline ( S4 Fig ) to determine:', '( i ) the chromosome copy number based on coverage depth ,', '( ii ) the genotype at SNP positions , requiring the determination of thresholds to call for homozygosity or heterozygosity ( S5 Fig ) ,', '( iii ) the extent of LOH , and', '( iv ) , the frequency , nature and location of the recombination events in individual strains , using the CrossOver algorithm from the ReCombine program , created for the analysis of tetrad data [27 , 28] .', 'First , we examined the sequence coverage among the individual chromosomes ( S6 Fig and S3 Table ) .', 'Remarkably , all genomes are euploid , indicating that chromosome segregation in the RTG process was accurate .', 'Nevertheless , two strains ( RTG4-S and RTG17-D ) displayed a coverage depth variation along two different chromosomes ( chromosomes V and XVI for RTG4-S and chromosomes III and V for RTG17-D ) , revealing in both cases a large duplication and a deletion of over 100 kb ( S6 and S7A Figs ) .', 'The duplication/deletion breakpoints , characterized using the Control-FREEC software [29] , are located near the Ty elements of the SK1 chromosomes [30] that are absent in the S288c chromosomes ( indicated on S7B Fig for RTG4-S ) .', 'Molecular validation by Pulsed Field Gel Electrophoresis and Southern blot analysis for the RTG4-S ( S7C Fig ) , suggests that these chromosomal-terminal Gross Chromosomal Rearrangements result from Break Induced Replication initiated between Ty elements located on different chromosomes ( S7D Fig ) .', 'The genotype at all SNP positions of the six RTG diploids isolated by selection is shown in Fig 2 . In each strain , the vast majority ( on average 86 . 3% ) of the SNP positions remained heterozygous as in the parental strain .', 'However , a substantial fraction ( on average 13 . 7% ) of SNP positions are homozygous for either parental origin ( Fig 2 , S4 Table ) , demonstrating that the RTG strains are recombined .', 'Remarkably , the total amount of polymorphisms exhibiting LOH varies from 15 . 2 to 27 . 8% between the RTG strains , demonstrating that the RTG process generates a high degree of genetic diversity .', 'Next , we analyzed the segregation at all SNP positions in the 15 pairs of mother-daughter RTG strains .', 'Since the RTG strains remained diploid , the genotyping of RTG pairs provides tetrad-like information concerning the segregation pattern of the four chromatids derived from of a single meiotic cell that underwent RTG .', 'On average , we observed that for 98 . 6% of the SNP positions , the genetic information segregated 2:2 in mother and daughter RTG pairs .', 'Among them , 89 . 2% carry a heterozygous genotype in both mother-daughter cells , as the parent diploid .', 'Conversely , 10 . 8% of the SNP positions segregating as 2:2 carry a homozygous genotype with opposite parental alleles in the mother and daughter cells ( S8 Fig and S4 Table ) .', 'This is exemplified in Fig 3 in which the genotype of the mother strain ( RTG11-M ) contains 16 . 3% of homozygous SNP positions , with 10 . 3% from S288c and 6% from SK1 , while the genotype of the daughter strain ( RTG11-D ) , contains 16 . 2% of homozygous SNP positions , but with the reverse percentage of parental alleles: 5 . 7% S288c and 10 . 3% SK1 .', 'The homozygous SNP positions exhibiting a 2:2 segregation pattern , grouped as tracts with reciprocal genotypes , correspond to LOH events resulting from reciprocal exchanges between non-sister chromatids .', 'Thus , the meiotic cell that exits from meiosis ( i . e . the mother cell ) inherits two non-sister chromatids and the bud ( i . e . the daughter cell ) inherits the other two non-sister chromatids , as expected from a successful re-entry into mitosis in the absence of DNA replication .', 'The non-sister chromatids are often but not always recombined .', 'As observed for the selected RTG strains ( Method 1 , see Fig 1 ) , the absolute frequency of acquired homozygosity is very different from one RTG pair to another .', 'In this dataset of 15 RTG pairs , we observed 136 reciprocal LOH tracts ( rLOH ) ( S5 and S6 Tables ) , with a wide variation , from 1 to 34 tracts per RTG pair .', 'On average , the rLOH tracts are large ( 141 kb ) , and in some cases , they cover most of the chromosome arm .', 'Most of the remaining SNP positions ( 1 . 4% ) exhibit a 3:1 segregation pattern of the genetic information , and carry a heterozygous genotype in one cell and a homozygous genotype in the other cell .', 'The homozygous SNP positions exhibiting a 3:1 segregation pattern are grouped into tracts with non-reciprocal genotype , corresponding to gene conversion .', 'We found 913 non-reciprocal LOH tracts ( nrLOH ) ( S5 and S7 Tables ) , from 5 to 139 events per RTG pair , which on average are small ( 2 . 3 kb ) compared to rLOH .', 'Finally , we also identified a low number of SNP positions that exhibited a 4:0 segregation pattern ( 0 . 06% ) , distributed in 38 tracts ( 1 to 5 per RTG pair ) .', 'These events can arise from multi-chromatid gene conversion events or from mitotic recombination events that occurred prior to meiotic induction .', 'The number of regions exhibiting LOH ranged from 5 to 87 per RTG strain , with sizes varying between 5 bp to 0 . 7 Mbp ( Fig 4A ) .', 'Overall , among the 36 RTGs ( 6 single and 15 mother-daughter pairs ) , 90% of the SNP positions were involved in at least one LOH event ( Fig 4B ) .', 'On average , 12 . 2% of the parental hybrid genome shows LOH , ranging from 0 . 3% ( RTG13-M/RTG13-D pair ) to 26 . 4% ( RTG7-M/RTG7-D pair ) .', 'The ratio of parental alleles also varies from one RTG to another ( Fig 4C ) .', 'Among the 10% of SNP positions that failed to exhibit LOH in all RTG strains , the vast majority are located around the 16 centromeres [15 , 31–34] .', 'Thus , the centromere-linked SNP positions always remain heterozygous after the equational segregation .', 'This is likely attributable to the depletion of meiotic DSB formation and recombination in the vicinity of the centromeres [15 , 31–34] .', 'To be noted , the maintenance of heterozygosity in the centromere region and in numerous locations along the chromosome arms eliminates the possibility that the LOH event resulted from iso-chromosomal non-disjunctions or reductional division .', 'The acquisition of the genome-wide recombination profile of the RTG-M and–D pairs provides unprecedented information on the nature of the recombination events ( gene conversions and/or crossovers ) .', 'Due to the occurrence of a single equational division that occurs when the cells exit from the prophase-I of meiosis resulting in RTG diploid cells , the method to detect the gene conversion and crossovers by genotyping is different than in the four haploid spores derived from a meiotic tetrad [2 , 19 , 28 , 33 , 35–39] .', 'The expected outcome of a single meiotic DSB repair by gene conversion and/or a crossover in a RTG pair is illustrated in Fig 5 .', 'DSB repair event by gene conversion is detected by a 3:1 segregation pattern of the SNP positions in the pair of RTG strains and is manifested by a non-reciprocal LOH ( nrLOH ) .', 'Differently , a crossover is detected by the occurrence of reciprocal tracts of LOH ( rLOH ) in the RTG pair , where the SNP positions segregation pattern is 2:2 .', 'The bioinformatics pipeline developed to detect gene conversions and/or crossovers events in diploid strains is shown in S9 Fig . To estimate the number of crossovers per RTG , we analyzed the SNP positions segregation pattern in the 15 mother and daughter RTG pairs .', 'The tracts of homozygous SNP positions that define the rLOH regions are comprised of two subclasses illustrated in Figs 3B and 5:', '( i ) the terminal rLOH ( trLOH ) , in which one end likely corresponds to the crossover site and the homozygosity extends to the end of the chromosomal arm ( formally to the ultimate SNP position ) , and', '( ii ) interstitial rLOH ( irLOH ) , where both ends of the homozygous tract are flanked by heterozygous tracts , thus reflecting the occurrence of two consecutive crossovers on the same chromosomal arm .', 'The double crossover can involve 2 , 3 or 4 chromatids , which is not distinguishable in diploid genotyping .', 'Altogether , we observed 70 trLOH and 66 irLOH ( S6 Table ) .', 'Assuming that each trLOH reflects the occurrence of one crossover and each irLOH reflects two crossovers , we detected a total of 202 COs in total , ranging from 1 to 54 COs per mother-daughter pair .', 'Concerning the frequencies of gene conversion events ( GC ) , we found that 1 . 4% of the SNP positions exhibited a 3:1 segregation pattern , leading to non-reciprocal tracts of LOH ( nrLOH ) as illustrated for the RTG11-M and -D strains in Fig 3 .', 'Altogether , among the 15 RTG mother-daughter pairs , we identified a total of 913 nrLOH tracts ( mean length of 2 . 3 kb ) , varying from 5 to 139 events per pair ( S8 Fig and S7 Table ) .', 'Once again , the nrLOH tracts can be interstitial or terminal .', 'Not surprisingly , the vast majority of the nrLOH is interstitial ( 847/913 = 93% ) , and corresponds to gene conversions , the canonical product of meiotic DSB repair by homologous recombination .', 'We observed that 164 interstitial nrLOH were located at the boundary of rLOH events , reflecting crossovers associated with gene conversions , while the remaining 683 are independent of rLOH events , reflecting NCOs .', 'The terminal nrLOH events ( 66/913 ) may be true terminal nrLOH events or may be interstitial if they ends in the non-genotyped repeated sub-telomeric regions of the chromosomes .', 'These terminal nrLOH events may result from Break-Induced replication ( termed terminal NCO or terminal gene conversion [2 , 40 , 41] ) .', 'Thus , among the 15 RTG pairs , we detected a total of 951 recombination events: 202 COs , including 164 COs associated with GC ( 81% ) and 38 COs not associated with a GC ( 19% ) , and 749 NCOs ( GC not associated with a detectable CO ) .', 'Due to the random segregation of the non-sister chromatids during the equational RTG division , additional COs may remain undetected upon SNP positions genotyping .', 'As illustrated in Fig 5 , upon equational segregation , a single CO leads to rLOH distal to the CO site in only half of the cases in mitotically growing cells , and therefore remains undetected in half of the cases [10 , 42] , while a GC leads to nrLOH regardless of the chromatid segregation .', 'Consistently , all NCOs will be detected as independent nrLOH , while , according to the chromatid segregation , half of the GC associated with a CO ( 81% of observed COs ) will be detected as such ( nrLOH at a boundary of a rLOH ,', 'i . e a GC associated with a CO ) , and half will be detected as an independent nrLOH ( NCO , or GC not associated with a detectable CO ) .', 'However , as illustrated in S10 Fig , the probability of CO detection is dependent on the number of CO per chromosome arm; It gradually increases from ½ to ⅔ when more COs occur on the same chromosomal arm .', 'Hence , assuming a random chromatid segregation pattern , and depending on the distribution of CO per chromosome arm , we expect that between ½ and ⅓ of the COs should remain undetected because they do not manifest as a rLOH event .', 'As well , the number of COs will also affect their distribution leading to interstitial or terminal LOH; as the number of COs increases , the probability of interstitial rLOH increases compared to that of terminal rLOH ( S10 Fig ) .', 'Taking into account these parameters , we estimate that the real number of CO in all 15 pairs ranges between 404 ( 202÷½ ) and 303 ( 202÷⅔ ) .', 'Since approximately 81% of the observed COs are associated with a GC , the corrected number of CO associated with a GC might range between 327 ( 404x0 . 81 ) and 245 ( 303x0 . 81 ) , and therefore the number of NCO ranges between 586 ( 913–327 ) and 668 ( 913–245 ) .', 'Altogether , this leads to an excess of NCOs over COs of 1 . 45-fold ( 327/404 ) to 2 . 21 fold ( 245/303 ) , a ratio opposite to the outcome of uninterrupted meiosis ( see Discussion ) .', 'To confirm the existence of these masked COs , we induced the sporulation of four RTG pairs ( RTG7M-D , RTG8M-D , RTG9M-D , RTG10M-D ) showing various extent of recombination frequencies ( S8 Fig ) and sequenced all four spores arising from one tetrad each .', 'As an example , the genotype of the RTG10-M and RTG10-D pair is illustrated in S11A and S11B Fig and the corresponding tetrads in S11C and S11D Fig . The SNP positions exhibited an expected Mendelian segregation pattern: the homozygous SNP positions of the RTG parental strain segregate 4:0 in the corresponding tetrad ( 99 . 69% ) , and the heterozygous SNP positions exhibit a 2:2 –or , occasionally , a 3:1 –segregation pattern ( 99 . 72% ) , validating our bioinformatics pipeline of diploid cell genotyping .', 'As anticipated , we identified several masked crossovers present in the parent RTG that are revealed in the RTG tetrad by the presence of 2 pairs of reciprocal recombinant molecules among the four spores of the tetrad ( S11C and S11D Fig ) .', 'Altogether , this revealed 37 masked COs ( ranging from 0 to 16 per RTG strain ) that did not lead to rLOH ( S11E Fig ) , in addition to the 77 COs leading to rLOH , which corresponds to the observed detection frequency of 77/ ( 37+77 ) = 67 . 5% , not significantly different from the expected ratio of 60 . 5% calculated from the distribution of CO per chromosome arm detected in those 4 RTG pairs ( p-value = 0 . 22 , Fisher exact test , S8 Table ) .', 'Two other observations should be mentioned .', 'First , we observed that 86% ( 32/37 ) of the masked COs are associated with an adjacent GC .', 'Association with GC is not statistically different between the detected ( 81% ) and masked COs ( 86% ) ( p-value = 0 . 75 , Fisher exact test ) .', 'Second , we observed that in each RTG pair , the number of masked COs is quantitatively related to the number of COs readily identified; namely , in RTG7M-D , RTG8M-D , RTG9M-D , RTG10M-D respectively , we detected 54 , 8 , 3 and 12 COs by LOH analysis and , we identified 27 , 2 , 1 and 7 masked COs upon tetrad sequencing .', 'Altogether , these results suggest that the detected and masked COs do not mechanistically differ but simply reflect the way the sister chromatids mitotically segregate upon RTG .', 'In the absence of recombination between the centromere and the mating type locus on chromosome III , the RTG strains remain heterozygous MATa/MATα , making it possible to repeat the RTG process .', 'Indeed , by phenotypic analysis of the mating behavior of the RTG strains , coupled with bioinformatic analyses , we found that 32/36 RTG strains were MATa/MATα , while the others were homozygous at the MAT locus , as MATa/MATa ( RTG11-M and RTG15- M ) or MATα/MATα ( RTG11-D and RTG15-D ) ( S2 Table ) .', 'Consistently , the MAT heterozygous ( MATa/MATα ) RTG strains sporulated while the MAT homozygous strains did not .', 'The rarity of exchanges between the centromere and the MAT locus on chromosome III is consistent with the unusually low frequency of meiotic DSB formation in this ~100kb region [15 , 43] .', 'To examine the genome dynamics of the RTG strains throughout successive passages of RTG , we conducted a RTG pedigree analysis starting from the RTG8-M strain and induced two additional rounds of RTG events , using the micromanipulation method and determined the cell genotype by WGS .', 'The genotype of the 10 RTG strains generated in this lineage , which all remained diploid , is shown in Fig 6 and S9 Table .', 'As expected , the LOH regions acquired at passage n were present in passage n+1 but additional LOH events appeared , indicating that these recombinant RTG strains retained the capacity to recombine and faithfully perform the RTG process .', 'Remarkably , as shown in one example in the lineage RTG8-M>RTG8-MD>RTG8-MDD , the genome homozygosity levels increased from 9 . 7% of the SNP positions ( 36 LOH tracts , including 6 reciprocal ones ) in passage 1 to 32 . 2% of the SNP positions ( 81 LOH tracts , including 17 reciprocal ones ) in passage 2 , finally reaching 46% of the SNP positions ( 117 LOH tracts , including 21 reciprocal ones ) in passage 3 .', 'In conclusion , the reiteration of the RTG protocol perpetuates the newly acquired LOH regions in a clonal manner , increases the degree of homozygosity and expands haplotype combinations in an incremental manner from one passage to the other .', 'Overall , extensive mosaic genomes of either parental origin are generated , a feature that raises the question of the potential roles of the RTG process in yeast genome evolution .', 'The genomic diversity of the recombinant RTG yeast cells has the potential to translate into phenotypic variations .', 'The SK1 parent is prototrophic for leucine and methionine , but auxotrophic for histidine while the S288c strain is auxotrophic for all three traits .', 'By comple
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['In somatic cells , recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events ( LOH ) , allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection .', 'However , inter-homolog recombination in somatic cells is rare , thus reducing potential genetic variation .', 'Here , we explored the property of S . cerevisiae to enter the meiotic developmental program , induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth , a process known as Return To Growth ( RTG ) .', 'Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome , derived from either parental origin .', 'Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous ( LOH ) events of various lengths , varying between a few nucleotides up to several hundred kilobases .', 'Furthermore , we show that reiteration of the RTG process shows incremental increases of homozygosity .', 'Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci ( QTLs ) in diploid strains without undergoing sexual reproduction .']
['The genetic diversity of eukaryotes relies on the diversification of the parental information , mostly occurring by recombination during gamete formation .', 'Homologous chromosomes also recombine in somatic cells , though much less frequently .', 'Here , we sequenced the genome of S . cerevisiae hybrid diploid cells that enter the processes of meiosis and Return To mitotic Growth ( RTG ) .', 'Remarkably , the RTG cells contain recombined diploid genomes derived from both parental origins .', 'Each RTG cell is diversely recombined both in terms of the frequency and location , with important implications in genome evolution of the species .', 'The generation of a diversely recombined diploid cell population has useful downstream genetic applications .']
['meiosis', 'cell cycle and cell division', 'variant genotypes', 'cell processes', 'chromatids', 'genetic mapping', 'dna', 'homologous recombination', 'chromosome biology', 'biochemistry', 'cell biology', 'nucleic acids', 'heredity', 'genetics', 'biology and life sciences', 'dna repair', 'dna recombination', 'chromosomes']