Why are allopolyploid hybrids sterile

There are mechanisms, however, by which the problem of hybrid sterility can be circumvented. This change in ploidy enables the correct pairing of chromosomes at meiosis and a diploid-like behaviour of the meiotic cell. Much like the viable interspecies zygotes of higher organisms, the zygotes produced by closely related Saccharomyces species form vegetatively proliferating populations of alloploid cells equivalents of somatic cells.

If hybridization is followed by genome doubling or the mating cells are diploid rare occasion , the hybrid will be allotetraploid and able to form viable ascospores possessing allodiploid chromosomal sets Naumov et al. Thus, the allotetraploid Saccharomyces hybrids can perform reductional division producing cells analogous to allodiploid gametes of allotetraploid plants and animals. However, there is an important difference between the allodiploid gametes and the allodiploid yeast spores.

Unlike the gametes that fuse in the course of fertilization restoring allotetraploidy or die, the allodiploid yeast spores germinate and produce vegetatively propagating allodiploid cells instead of fusing. Later, when appropriate signals arrive from the environment e.

Thus, in spite of the viability of their first-generation F1 spores, the allotetraploid Saccharomyces hybrids can also be considered sterile. This kind of sterility is referred to as F1 sterility to distinguish it from the sterility of the allodiploid hybrids Sipiczki, Many Saccharomyces strains participating in beer and wine fermentation are alloploids or at least display traces of interspecies hybridization event s.

The other brewing yeast taxon, Saccharomyces bayanus S. Alloploids are far less common among wine yeasts. As the progenitors are not known, it is practically impossible to reconstruct these events.

However, the examination of synthetic hybrids produced under laboratory conditions from genetically characterised parental strains may provide an insight into the postzygotic development of the alloploid genome.

To test the applicability of this approach, we produced a synthetic S. We observed various structural interactions between the component genomes and gradual elimination of genes and chromosomes from the S. These findings and the results of the molecular analysis of natural chimeras lead to a model for gradual stabilization of the Saccharomyces alloploid genomes Sipiczki, Although consecutive meiotic divisions were proposed to be a major driving force for the stabilization process, the model did not address how the hybrid overcomes sterility.

In this work, we show by analysing a large number of S. The resulting fertile alloaneuploid F1 segregants nullisomic for Chr. To the best of our knowledge, this is the first report proposing a mechanism for breaking down postzygotic barrier between Saccharomyces species.

The restoration of fertility and the subsequent genome reduction provide possibilities for genome rearrangements leading to alloaneuploids and mosaics whose genomes consist of nearly complete genomes from one hybridising partner and certain genes from the other partner horizontal gene transfer.

Saccharomyces strains used in this study are listed in Table 1. Synthetic interspecies hybrids were produced by mass-mating of S. To test the mating activity of nonsporulating cultures, dense suspensions of cells were mixed with suspensions of mating-type tester strains and samples of the mixed suspensions were dropped on plates of sporulation medium.

After 5 days of incubation, spore formation was checked microscopically. The mating activity of sporulation-proficient segregants was tested by mass-mating of their sporulating cultures with cells of the S. As the segregants and the testers had different auxotrophic markers, the production of prototrophic colonies was taken as evidence of mating capability.

For random spore analysis, samples were taken from the sporulating cultures, treated with zymolyase the concentration of the enzyme and the duration of treatment varied according to the sensitivity of the ascus walls , sonicated to separate the ascospores and plated out on YPGA plates.

Ascus dissection by micromanipulation, isolation of tetrads of spores and the analysis of the phenotypes of the spore clones were performed as previously described Antunovics et al. PCR parameters are described in the references given in Table 2. The amplified DNA fragments and the subfragments generated by endonuclease treatments were identified and separated by electrophoresis in agarose gel.

The method of electrophoretic karyotype analysis was described by Antunovics et al. The hybridization probes were labelled with the DIG-High Prime system Roche and used for hybridization to the membranes of the karyotypes according to the manufacturer's instructions. The primers and the PCR parameters used for amplification of chromosomal sequences probes for Southern hybridization are described in Table 2. The S. Plasmids were isolated from 20 transformed bacterial colonies and used to transform the leucine auxotrophic S.

The identity of the inserted fragments was checked by sequencing their ends. The stability of the transformants was tested by culturing their cells in the nonselective complete medium YPGL. The colonies that had lost the transforming plasmid did not grow on MMA.

Saccharomyces cerevisiae and S. In mixed populations, their cells and spores can interact in many ways including sexual mating resulting in interspecies hybrids. To simulate natural situations, we mixed cultures of S. Then we plated out samples on a selective minimal medium to identify prototrophic hybrids. Fifty-seven prototrophic colonies were isolated from the plates and deposited in a freezer to prevent genetic changes that might occur during vegetative propagation of the hybrid cells.

The isolates were then tested for temperature sensitivity and melibiose fermentation S. To verify their hybrid nature, we subjected randomly selected hybrids and the parental strains to electrophoretic karyotyping. As shown in Fig. Electrophoretic karyotypes of the parental strains and four synthetic hybrids. Chromosome numbering is shown on the left size for S. The numbering is based on the reference Antunovics et al.

Each hybrid was tested for sporulation by culturing on sporulation medium and for spore viability by random spore analysis Table 3. Segregation of auxotrophic markers in random spore analysis was taken as evidence of production of viable spores.

The rest of the hybrids produced more spores and could thus be subjected to random spore analysis. None of them could conjugate with the mating type testers listed in Table 1. Seventy per cent of the hybrids were not sterile because they produced auxotrophic segregants. We hereafter call the viable spores of the hybrids F1 spores and refer to the clones of vegetative cells produced by them as F1 clones.

Hybrids and their offspring analysed in detail. Tetrads and spore clones with black background were used for production of filial generations. Percentages of viable spores are shown in brackets. Sporulation of hybrids and segregation of auxotrophic markers in random spore analysis. However, the spores of the H21 and H29 F1 clones were all dead. Thus, these hybrids were F1-sterile. In contrast, certain F1 clones of the hybrids H10 and H35 produced viable F2 spores, indicating that these hybrids were not fully F1-sterile.

From one of the F2 spore clones, we isolated tetrads of F3 spores and found high levels of spore viability. Then, we isolated spores from two F3 clones to produce F4 generation. The F4 clones obtained also sporulated efficiently. The genealogy of the spore clones and the spore viability in selected clones are shown in Fig. To explore the genomes of the hybrids and their descendents, we tested the spore clones for the presence of parental markers and chromosomes. The eleven markers covered eight chromosomes of the S.

As no 1 : 3 and 3 : 1 tetrads were found, we presumed that the wild-type S. Both categories were tested for mating activity with S. In brackets: the chromosomal location of the gene in the Saccharomyces cerevisiae genome. Fragment size. The four hybrids analysed were heterozygous for all molecular markers Table 4.

Their descendents also possessed both parental orthologues of most genes. In a few spore clones, the S. These results confirmed that the entire Chr. The copy number of the leu2 allele can be higher in the hybrid when the S.

III of the S. To verify the absence of a S. In our previous study on a similar synthetic hybrid Antunovics et al. In those clones, the S.

Remarkably, the HIS4 orthologue is also located on scaffold s10 in the S. To ascertain whether this scaffold corresponded to the band that was missing in the karyotypes of the fertile F1 clones, we amplified the S.

As presumed, the probe reacted with Chr. Probing the karyotypes with a labelled S. The only exception was Chr. III which does not bind the probe Antunovics et al. However, certain spore clones differed in signal intensity at the poorly resolved group of the largest chromosomal bands Fig. By modifying the running parameters of electrophoresis, we managed to separate the bands and found no segregation Fig. Chromosomal patterns and hybridisation with Saccharomyces cerevisiae -specific subtelomeric probe in hybrids and F1 descendents.

One example of tetrads is shown for each hybrid. Arrowheads mark Saccharomyces uvarum Chr. Hybridisation of labelled LEU2 to chromosomal bands. Lane numbering is as for Fig.

Arrowhead with double line: Saccharomyces uvarum Chr. Arrowhead with single line: Saccharomyces cerevisiae Chr. Separation of large chromosomes and hybridization with Saccharomyces cerevisiae -specific subtelomeric probe.

Numbering of S. To reveal further differences between the genomes of the hybrids and their descendants, we performed RAPD analysis with two primers and amplified interdelta sequences Table 4. There were only two S. The amplification of interdelta sequences revealed higher diversity. No fragments were amplified from the S. The inability of the S. As Chr. To test whether a similar interaction may also take place between the MAT loci of the hybridised species, we cloned the MAT cassettes of the S.

We obtained both conjugating and nonconjugating transformants. The sterility of the nonconjugating transformants was unstable.

The sterility of the transformants and the simultaneous loss of their prototrophy and sterility demonstrated that a S. Using the mass-mating method based on complementation of auxotrophic markers, we generated 57 novel synthetic hybrids of S. As the mating mixture contained both spores and vegetative cells, interspecies hybrids could arise from all possible combinations of spores and vegetative cells of the partners.

These combinations were unlikely to produce hybrids of identical ploidy because spores have smaller genomes than vegetative cells. In one case, transgene silencing occurred more frequently in Arabidopsis thaliana tetraploids than in A. However, several factors cannot be ruled out in the observation of this phenomenon, including duplication of the strong 35S promoter from cauliflower mosaic virus in the transgene.

Unfortunately, the generality of this change could not be determined because multiple independent autopolyploids were not examined.

Conversely, extensive evidence for epigenetic remodeling is available in allopolyploids. Structural genomic changes, such as DNA methylation , and expression changes are reported to accompany the transition to alloploidy in several plant systems, including Arabidopsis and wheat Shaked et al.

The most detailed information is available for the model system Arabidopsis. For instance, in a cross of A. Taken together, these results suggest that the instability syndrome of neoallopolyploids may be attributed primarily to regulatory divergence between the parental species, leading to genomic incompatibilities in the allopolyploid offspring. Aneuploidy might also be a factor in epigenetic remodeling in neoallopolyploids, either by altering the dosage of factors that are encoded by chromosomes that have greater or fewer than the expected number of copies leading to changes in imprinted loci, or by exposing unpaired chromatin regions to epigenetic remodeling mechanisms.

In the latter case, this susceptibility of meiotically unpaired DNA to silencing was first reported for the fungus Neurospora crassa , but it appears to be a general phenomenon. Therefore, some of the epigenetic instability that is observed in allopolyploids might result from aneuploidy.

At first sight, the epigenetic changes observed in polyploids would seem to be deleterious because of their disruptive effects on regulatory patterns established by selection. However, these epigenetic changes might instead increase diversity and plasticity by allowing for rapid adaptation in polyploids. One example may be the widespread dispersal of the invasive allopolyploid Spartina angelica.

However, it is not clear whether the success of this species can be attributed to fixed heterosis or to the increased variability that results from epigenetic remodeling. Polyploidy is also believed to play a role in the rapid adaptation of some allopolyploid arctic flora, probably because their genomes confer hybrid vigor and buffer against the effects of inbreeding.

However, fertility barriers between species often need to be overcome in order to form successful allopolyploids, and these barriers may have an epigenetic basis. Recent studies have provided interesting insights into the regulatory and genomic consequences of polyploidy. Together with the emerging evidence of ancestral duplication through polyploidization in model plant, fungus, and animal species, knowledge of these consequences has stimulated thinking about the relationship between early polyploidization events, the success of the polyploidy, and the long-term fate of new species.

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Plant Cell 5 , Mittelsten Scheid, O. A change of ploidy can modify epigenetic silencing. Proceedings of the National Academy of Sciences 93 , Shaked, H. Sequence elimination and cytosine methylation are rapid and reproducible responses of the genome to wide hybridization and allopolyploidy in wheat.

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Chromosomal Abnormalities: Aneuploidies. Chromosome Abnormalities and Cancer Cytogenetics. Copy Number Variation and Human Disease.

Genetic Recombination. Human Chromosome Number. Trisomy 21 Causes Down Syndrome. X Chromosome: X Inactivation. Chromosome Theory and the Castle and Morgan Debate. Developing the Chromosome Theory. Meiosis, Genetic Recombination, and Sexual Reproduction. Energy Flow 3. Carbon Cycling 4. Climate Change 5: Evolution 1. Evolution Evidence 2. Natural Selection 3. Classification 4.

Cladistics 6: Human Physiology 1. Digestion 2. The Blood System 3. Disease Defences 4. Gas Exchange 5. Homeostasis Higher Level 7: Nucleic Acids 1. DNA Structure 2. Transcription 3. Translation 8: Metabolism 1. Metabolism 2.