The genus Saccharomyces comprises eight „natural species”, namely S. arboricola, S. cerevisiae, S. eubayanus, S. jurei, S. kudriavzevii, S. mikatae, S. paradoxus, and S. uvarum (recently reviewed by Alsammar and Delneri1) and many strains of chimeric (admixed) genomes that are, somewhat superficially, also called „interspecies hybrids”. Two groups of the chimeric, mostly brewing strains of highly diverse genome structures are accommodated in the so-called „hybrid species” S. bayanus and S. pastorianus (S. carlsbergensis) (e.g. 1–5). The chimeric strains identified in other environments (e.g. in wine-making processes) are not grouped in separate species and are assumed to have evolved from hybrids of natural species by loss and rearrangements of mosaics in the parental subgenomes (for a review, see e.g. 6).
The taxonomic division of the genus was mainly based on the biological species concept and later confirmed by the analysis of barcode and genome sequences. In the biological species concept introduced to Saccharomyces taxonomy by Naumov7, the species are populations of interbreeding strains isolated by sterility barriers. While conspecific strains form fertile hybrids (producing functional gametes), the strains that belong to different species either do not form hybrids (prezygotic sterility barrier) or their hybrids do not form functional gametes (ascospores) (postzygotic sterility barrier). The Saccharomyces species are isolated by postzygotic sterility barriers. All Saccharomyces species can form viable (allodiploid) hybrids with any other Saccharomyces species but the hybrids either do not sporulate or their spores are not viable.
Allodiploid sterility is mainly due to the failure of the chromosomes of the subgenomes to pair in meiosis I (e.g. 8–11) which results in the abruption of the meiotic process (“first sterility barrier”). In plants, allodiploid sterility can be circumvented by genome duplication, which „diploidises” the subgenomes (for a review, see 12). In the autodiploid subgenomes of the allotetraploid plant hybrid, each chromosome has a homologous partner to pair with, which allows successful meiosis. The allodiploid plant gametes produced in the allotetraploid meiosis are functional and can mate with other gametes to form allotri-, allotetra- and even allopolyploid zygotes (hybrids) depending on the ploidy of the partner gamete13.
Genome duplication also occurs in yeast interspecies hybrids14–17 and the resulting allotetraploid hybrids also produce viable allodiploid gametes (ascospores capable of germination). Their viability is frequently misinterpreted as the breach of the sterility barrier by genome duplication18. However, in contrast to the allodiploid gametes of the plant hybrids, the allodiploid ascospores cannot function as gametes14,19. This difference between the plant hybrids and the Saccharomyces hybrids is attributable to the different mechanisms of the regulation of sexual processes in plants and yeasts.
In Saccharomyces, the MAT cassettes in the MAT loci determine which of the alternative sexual programmes, mating-fertilisation or meiosis-sporulation, is active. In a haploid genome there is only one MAT locus, which contains either a MATa or a MATalpha cassette. Single copies of MAT cassettes allow mating but repress meiosis-sporulation. Two haploid cells having different cassettes in their MAT loci can mate (conjugate) and form a heterozygous MATa/MATalpha diploid. MATa/MATalpha heterozygosity blocks the mating programme and mating-type switching but allows meiosis20. However, in spite of the activation of the meiotic programme, the allodiploid cells are prevented from producing viable and functional haploid ascospores (gametes) by the first sterility barrier. If the allodiploid genome of the hybrid becomes allotetraploid by spontaneous genome duplication, it will be able to produce viable allodiploid ascopores like the allotetraploid plants, however, in contrast to the plant allodiploid gametes, the yeast allodiploid ascospores are sterile. They cannot mate because of their MATa/MATalpha heterozygosity (“second sterility barrier”)19. The two sterility barriers (the double sterility barrier) ensure the reproductive (biological) isolation of the Saccharomyces species. Due to the second sterility barrier, which has no counterpart in plants, the interspecies Saccharomyces hybrids remain sterile even upon whole-genome duplication.
Because of their sterility, the alloploid hybrids can mate neither with the parental strains nor with strains of other species. Thus, allodiploid sterility prevents both introgressive backcrosses with parental strains and hybridisation with a third species. To overcome this obstacle, two “natural”, non-GMO strategies have been proposed for hybridising of a two-species hybrid with a third species21.
One strategy is based on the occasional breakdown of the second sterility barrier by the loss of MAT heterozygosity due to occasional inaccurate distribution of chromosomes during allotetraploid meiosis. If a spore receives a MAT-carrying chromosome only from one of the subgenomes (los of MAT-heterozygosity), it becomes mating-competent. In this alloaneuploid spore (nullisomic for the MAT-carrying chromosome in one of the subgenomes) the mating programme is released from repression14. This spore can mate with a haploid cell of opposite mating activity, but the hybrid is not triploid but only aneuploid (segmental triploid), nullisomic for the chromosome lost during meiosis. Since additional parental chromosomes can also be lost during meiosis, these hybrids only have mosaic genomes consisting of aneuploid subgenomes15. If the mating partner is a cell of a third species, the hybrid will be a segmental allotriploid (alloaneuploid). The second sterility barrier can also be overcome by integrating genetically modified drug-inducible HO genes (HO codes for a mating-type switching endonuclease) in the genomes of the parental strains before hybridisation. The “artificial” (drug-induced) expression of these genes reactivates the mating-type switching process normally repressed by the MATa/MATalpha heterozygosity and makes certain hybrid cells homozygous and thus mating-competent. However, the hybrids obtained in this way also had chimeric genomes consisting of mosaics of the parental genomes22. The failure to produce polyploids with alloeuploid genomes can be attributed to the instability of the hybrid genomes that can easily loose chromosomes during mitotic and meiotic divisions of the hybrid cells by the “postzygotic” processes designated GARMi and GARMe, respectively6. Thus, “true” allopolyploid hybrids possessing entire parental genomes cannot be produced with genetic manipulation of HO expression either.
The other natural strategy proposed in reference21 is based on the rare mating of sterile diploid cells. Gunge et al.23 described the phenomenon referred to as “rare mating” in S. cerevisiae. They noticed that in MATa/MATalpha S. cerevisiae diploid cultures, very rarely, certain cells escaped the block of the mating programme and conjugated with mating-competent cells or spores of other strains. In a previous study, we managed to make use of this phenomenon to hybridise sterile two-species kudvarum (S. kudriavzevii x S. uvarum) hybrids with S. cerevisiae strains to obtain three-species cekudvarum (S. cerevisiae x S. kudriavzevii x S. uvarum) hybrids without gene manipulation19. We found that the hybrids had repressed mating and mating-type switching programmes but we did not examine their genome structures. In the current work, we present data demonstrating that the three-species hybrids created in this natural way have euploid subgenomes consisting of complete parental sets of chromosomes. Since the hybrids do not form viable ascospores, their genomes are stable. This is the first report on constructing stable euploid three-species Saccharomyces hybrids. Interestingly, the mitochondrial genomes were uniparentally inherited.