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Direct mating

Classical direct mating (Fig. 5.3B), accomplished via controlled spore-to-spore mating, requires two parental strains that produce viable spores with high efficiency, a characteristic that is generally not found in brewing yeasts, especially not in lager yeasts. Ale yeasts vary in their spore viability, with some strains more capable of classical

’Natural’ mating-based strain improvement techniques

Figure 5.3 ’Natural’ mating-based strain improvement techniques. Sexual hybridization techniques are often used as a way to generate artificial diversity in yeasts that are capable of (1) mating with each other and subsequently going through successful meiosis and sporulation (e.g. genetically well-behaved yeasts within the same species, e.g. diploid strains of S. cerevisiae), or (2) mating with each other through the natural ability of cells or spores of opposite mating types to fuse with each other (e.g. between haploid or spore-producing strains of the various Saccharomyces species). Due to the sometimes complex genetics of yeast, different techniques have been developed. Most techniques start from two parental strains (i.e. the cells shown in the top row of each panel) that have been selected for target phenotype(s), and result in diploid or higher ploidy strains that can be selected or screened for the desired phenotype. The greyscale bar at lower left indicates the strength of the phenotype, for example, dark = strong ethanol tolerance, light = weak ethanol tolerance. (A) Standard mating. For ‘standard mating’, two haploid yeast strains of opposite mating type (‘a’ and ‘a’, respectively) and which are capable of continually asexually (mitotically) dividing as haploids (due to inactivation of the HO-endonuclease; see main text) can be pre-screened for desired traits. If selection for diploid cells is possible for this combination of strains, it is very simple to mix a large mass of cells from each parent strain together, whereupon cells of opposite mating type, when located close to each other, can fuse and form ‘a/a’ diploid cells (Fig. 5.1); these can then be evaluated for presence and strength of the desired trait. If there is no selection available for diploid cells, then individual cells from each parent can be microscopically manipulated to be adjacent to each other on a petri plate (at a marked location), and the colony appearing there will be composed of the diploid cells arising from the original mating of the two cells. This technique can be performed with haploid parents of the same species, or haploid parents of different species within the Saccharomyces genus. (B) Direct mating. When one or both of the parental strains contain an active HO gene, the strains are essentially always diploid and instead of standard haploid-haploid mating, direct spore-to-spore mating must be used. Two parental strains (shown as intermediate in phenotype strength, but in the case shown, are heterozygous) are each sporulated, then the haploid spores (before they can germinate and self-mate to form diploids) are microscopically manipulated to be placed adjacent to each other on a petri plate as described above; the diploid colony of cells arising from the mating of the two spores can then be seen on the plate and chosen for further evaluation. Because the parents are heterozygous, their spores are genetically diverse as shown by the differences in colouring, and randomly choosing spores for mating will result in a wide range of the phenotype of interest. However, if a screening or selection step is applied to the spores before mating, then only those spores with the desired phenotype can be chosen for mating, leading to enhancement of the phenotype; if a DNA-based screening of a single spore is used to identify spores with known desired gene variants, this method becomes a type of ‘marker-assisted breeding’. (C) Rare mating. For ‘rare mating’, strains are crossed without a sporulation step. This is possible because normal ‘a/a’ diploid yeasts will very rarely undergo a mating-type switch on one chromosome, yielding an ‘a/a’ or ‘a/a’ diploid cell. These cells can subsequently mate with a haploid cell of the opposite mating type as shown. It is important to note that rare mating is not limited to the development of triploid yeasts. For example, tetraploid hybrids can be obtained if the parent on the right was an ‘a/a’ diploid cell instead of an ‘a’ haploid. (D) Mass mating and genome shuffling. For ‘mass mating’, multiple parental strains, or a heterogeneous population (e.g. after mutagenesis) of the same parental strain, can be used. After mass sporulation and mixing of the resulting spores, mass mating will occur. These rounds of mass sporulation and mass mating can be repeated multiple times, a process which is one way to perform so-called genome shuffling. In genome shuffling, the mass-sporulation and mass-mating steps can also be replaced by protoplast fusion (Fig. 5.4B).

Other strain improvement techniques

Figure 5.4 Other strain improvement techniques. As in Fig 5.3, parental strains are shown in the top row, and the greyscale bar at lower left indicates the strength of the phenotype. (A) Cytoduction. Cytoduction (cell fusion without nuclear fusion) can be used to transfer cytoplasmically inherited traits, such as mitochondria or other organelles between cells. It can also result in transfer of single chromosomes between nuclei. The parental strain containing the desired cytoplasmic trait first needs its KAR1 gene inactivated (shown as karl-). Next, both parental strains are mated, if capable (if not, they can be fused by protoplast fusion). Because nuclear fusion (karyogamy) is blocked due to the karl - defect, the heterokaryon (cell containing two unfused nuclei) subsequently divides into cells containing a nucleus of only one parent but the cytoplasmic components of both parents (=heteroplasmons). With proper genetic selection, this technique can also yield so-called disomic strains that contain the full chromosome complement of one parent plus one chromosome from the other parent. (B) Protoplast fusion. For protoplast fusion, cell walls are first removed (usually by enzymatic means), after which the cells are asexually merged by incubation in osmotically supportive medium and in high concentration so cells are in close proximity. After fusion, the cell wall regenerates and the heterokaryons may undergo karyogamy to form asexually stable hybrids. (C) HO-induced mating and hybridization. This technique allows genetic analysis of a sterile or otherwise intractable strain via a tetraploid intermediate, as well as efficient production of Saccharomyces interspecific hybrids. Each parent strain is transformed with a plasmid containing an inducible HO gene, and because each plasmid has a different dominant selectable marker, this allows selection of successful mating events on double selective media. In the example shown here, parental diploids are mated to form a tetraploid, but other starting ploidies may be used.

breeding techniques; this must be established on a strain-by-strain basis, which can be a laborious process. Strains also vary in their sporulation efficiency, and sporulation conditions developed for laboratory strains might not be optimal for all brewing yeasts. For example, lager yeasts prefer lower sporulation temperatures than do laboratory S. cerevisiae strains (Bilinski et al., 1987), and sporulation conditions may thus need to be optimized on a strain-by-strain basis, again a laborious endeavour. However, once viable spores have been isolated, breeding lines for future mating experiments can be developed.

The direct mating approach has been carried out with brewing yeasts, either with strains that have high spore viability, or with rare surviving spores, as shown by the following examples. Gjermansen and Sigsgaard (1981) isolated rare viable spores of lager yeast and established breeding lines to develop strains with fermentation performances similar to the parental strains. Bilinski and Casey (1989) performed mating of rare viable S. pastorianus spores with S. cerevisiae ale strain spores, observing altered (and in some cases desired) fermentation characteristics. Similarly, Sanchez et al. (2012) improved the thermotolerance, osmotolerance, and ethanol tolerance of lager yeast by mating rare viable S. pastorianus spores with spores from S. cerevisiae strains derived from multiple sources. Steensels et al. (2014a) used direct mating to improve the aroma characteristics in ale yeast, but first screened out yeasts that were incapable of forming stable haploid breeding lines. Krogerus et al. (2015) mated spores from a natural auxotrophic mutant (ura-) S. cerevisiae ale yeast strain to spores from a natural auxotrophic mutant (lys-) S. eubayanus yeast strain, and selected for prototrophy, thus isolating novel lager yeast-type interspecific hybrids. Similarly, Mertens et al. (2015) also generated novel lager- type hybrids without underlying auxotrophic mutations, by directly manipulating spores from unaltered ale yeast strains to be adjacent to spores from unaltered S. eubayanus yeast strains, thus allowing fusion and mating without any selection steps. In both studies many of the novel lager-type hybrids showed promising unique fermentation properties and aroma profiles not seen in production lager strains.

A very recent study has characterized the whole genome sequences of over 100 S. cerevisiae ale and other brewing yeast strains (Gallone et al., 2016), yielding a rich trove of genomic data, including polymorphisms that are known or suspected to contribute to industrially relevant phenotypes. Such knowledge can allow the use of PCR or other DNA-based assays, on a fairly large scale, to select only those segregants or cells carrying the desired genetic variants for further breeding; this has been called ‘marker-assisted breeding' and has been used for many years in crop and livestock breeding. As a proof-of-concept for brewing yeasts, Gallone et al. (2016) created new S. cerevisiae intraspecific hybrids with altered aromatic properties using marker-assisted breeding.

In general, however, direct mating can be laborious and time-consuming, especially for strains, such as many brewing strains, that sporulate poorly. Furthermore, the direct mating strategy is a gamble: because brewing strains are generally heterozygous, meiotic segregation results in spores that can be very diverse genetically, so that some of the spores used in matings may carry inferior traits; also, it is extremely challenging to select for genes that are closely physically linked to loci that cause spore inviability. Thus, other hybridization methods have been developed that do not rely on the ability to generate viable spores, as follows.

 
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