End‐of‐fermentation analysis of BC‐hybrids The amount of viable cells (CFUs, compared to total cell count) was indicated as survival rate (A) and the percentage of petite cells of CFUs after end‐fermentation was determined. Petite cells, i.e. cells that could not use glycerol as carbon source, were determined by replica‐plating from YPD plates on YP glycerol plates. Data are averaged from two independent experiments. SEM is shown ( n > 6)

At the end of fermentation, yeast cells were cropped and their robustness was analysed by quantifying the percentage of cell survival and the rate of petite cell formation (Figure 3 ). Interestingly, BC1 and BC2 provided different results. The amount of viable cells at the end of fermentation was higher in BC2 than in BC1, yet both strains had an improved rate of viable cells compared to the lager yeast strain (Figure 3 A). The faster fermentation of the BC strains could thus be a consequence of the increased vitality/viability throughout fermentation. The amount of petite cells at the end of fermentation showed an average of ca. 4% petite cells for the lager yeast strain. This is within the normally observed range in lager yeast. BC1 performed less well in this respect, with an elevated count for petites. Remarkably, cells of strain BC2 cropped at the end of fermentation contained only a very small fraction of petites (Figure 3 B). Fermentation performance and analysis of parameters at the end of fermentation thus clearly showed the possibility for improvement of lager brewing yeast upon breeding with S. cerevisiae .

Lager yeasts show an increased resistance to oxidative stress. Representative fermentation kinetics of the BC1 and BC2 hybrid strains in comparison with a lager yeast strain. High gravity fermentation was done with YP maltose as carbon source adjusted to 25°Plato. Fermentation temperature was 22°C. Data are averaged ( n > 4). The plot contains reduction of sugar content as well as increase in weight loss (g/l) over time

Lager yeasts are more sensitive to high‐temperature and high‐salt stress than S. cerevisiae strains. Yeast strains were grown overnight in YPD with 2% glucose, washed with water and adjusted to an OD 600nm = 0.08, followed by 1:10 serial dilutions that were spotted on YPD plates supplemented with 10–12% ethanol or 1 m NaCl where indicated. Plates were incubated at either 30°C or 37°C (A, B). Representative pictures were taken after 5 days of growth. S. cerevisiae strains: BY laboratory yeast strains and a wine yeast strain. Lager yeast strains are commonly referred to as S. carlsbergensis . CG strains are lager yeast spore clones and BC strains are derived from crosses of B Y and C G strains

Lager yeast is adapted to low‐temperature fermentation conditions. We used several lager yeast strains from different origins to compare their sensitivity to high‐temperature, high‐salt and ethanol stress (Figure 1 A). S. cerevisiae strains, either laboratory yeast or wine yeast, were much more robust under these conditions than all lager yeasts, although within lager yeast strains different levels of sensitivity against high ethanol concentrations were observed. This is of interest, as lager yeasts may experience high osmotic stress at the beginning of fermentation and ethanol stress at the end of fermentation (Gibson et al ., 2007 ). Lager yeast hybrids are very poor sporulaters. To facilitate lager yeast breeding, we previously generated lager yeast spore clones with distinct mating behaviour and his4 , leu2 , ade2 auxotrophies (Gjermansen, unpublished). We made use of two of these strains that mated either as MAT a (CG1161) and MATα (CG1162) and mated them to the corresponding BY4742 ( MATα , ura3 ) or BY4741 ( MAT a , ura3 ) laboratory strains. Selection on SD – ade – ura plates was used to identify the backcross strains BC1 (BY4741 × CG1162) and BC2 (BY4742 × CG1161). The stress profile of BC1 and BC2 strains was compared with the parental strains and a lager yeast production strain (Figure 1 A, B). We found that a single backcross generated F1 hybrids that were clearly improved in their resistance profile over lager yeasts. To further characterize these new hybrids, we performed bench‐top fermentations using high‐gravity maltose medium (25ºPlato) at 22°C. We were especially interested in higher temperature fermentation performance instead of fermentations under lager brewing conditions (10–15°C), as we hypothesized that an increase in S. cerevisiae genome content would also improve the performance at higher temperatures, e.g. with respect to total time of fermentation. Fermentation curves of these F1 hybrids, i.e. sugar consumption and weight loss generated by CO 2 production, were compared to a S. carlsbergensis lager yeast strain (Figure 2 ). It was evident that under high‐gravity brewing conditions and at elevated temperature the new hybrids fermented maltose faster than lager yeast. This feature was stable upon repitching (not shown).

Mating‐type genotype in lager yeast spore clones may not correspond to mating behaviour

This encouraging result of strain improvement based on breeding lager yeast spore clones with S. cerevisiae promoted a more detailed study. Lager yeast is a hybrid between two Saccharomyces species. Usually, one parent is of S. cerevisiae origin while the other is closely related to S. bayanus (Libkind et al., 2011). Based on this hybrid nature lager, yeasts generally sporulate very poorly. Added to this is the uncertainty of the chromosomal content of spore clones (≥ 2n or allodiploid).

To disclose the MAT genotype and the actual mating behaviour of spore clones, we used PCR with MATa‐ and MATα‐specific primers (Huxley et al., 1990). This generated the somewhat surprising result that often lager spore clones were heterozygous at the MAT locus and yet showed a specific mating behaviour with tester strains (Figure 4a). This method does not distinguish copy numbers of MAT loci; thus, at present we do not know whether an imbalance at MAT or the presence of cerevisiae/non‐cerevisiae MAT‐loci generates this non‐canonical mating ability. In our limited study we found more a‐ than α‐maters in lager yeast spore clones of different origin. From a breeding perspective, we favour a strain in which mating behaviour is matched by MAT locus composition (Figure 4a).

Figure 4 Open in figure viewer PowerPoint Assessment of yeast strain mating type by colony PCR. Cells from the indicated strain were used for colony‐PCR, using three primers to amplify either a MATa‐ or MATα‐specific band. Equal amounts of these PCR reactions were separated on agarose gels. Ethidium bromide‐stained gel images are shown (A, B). Strains from (A) were further used in mating reactions against haploid tester strains. The respective results are shown as mating phenotype. The ale yeast spore clones were derived from four‐spored asci. Strains with both MAT loci are regarded as diploids. Whenever a single band was observed in the mating type PCR, it corresponded with the mating phenotype

In contrast to lager yeasts, ale and distiller's yeasts are generally non‐hybrid strains of S. cerevisiae. Thus, to generate a more robust lager yeast strain, we aimed at crossing lager yeast with an ale yeast to combine the properties of two experienced wort‐fermenting strains. To this end we also generated spore clones of an ale yeast strain (MATa/MATα) and analysed their MAT locus composition (Figure 4b). Also in this case we identified strains with both mating types (which could simply have been diploid cells) and selected only strains with consistent mating‐type and mating behaviour for further breeding (see Table 1 for list of strains). Mating properties of the ale isolates were stable and were confirmed by PCR and with mating to tester strains BY4741 (MATa) and BY4742 (MATα) (not shown).

Lager vs ale yeast hybrids were generated by mixing the strains and using micromanipulation to isolate zygotes (Figure 5A). Of the isolated cells, < 50% formed colonies. These were further analysed by PCR to identify strains that indeed carried both mating‐type loci (Figure 5B). Due to the appearance of odd‐looking cells, not all of the cells originally manipulated represented true hybrids. Lager yeasts generally can utilize melibiose (galactose‐glucose disaccharide), due to the presence of MEL genes. This feature was analysed by growth with the chromogenic substrate X‐α‐gal, which is converted to a blue dye in the presence of a secreted α‐galactosidase (not shown). The MEL+ phenotype combined with growth at 37°C can corroborate the generation of lager × ale hybrids. We employed a growth assay at 37°C to eliminate lager yeast cells and then verified the presence of the lager yeast S. bayanus copy of YBR033W, using specific primers (Figure 5C; Torriani et al., 2004). This, in combination with the MAT locus PCR, unambiguously identified 13 lager × ale hybrids (C600–C612). These new hybrids were tested for stress resistance against high temperature and high salt concentration (Figure 6). The parental strains segregated in phenotype according to lager and ale yeasts. Ale yeasts and their spore clones were generally more resistant to higher temperature and salt stress than lager yeasts and their derivatives. As seen with the previous backcrosses, BC1 and BC2 also in the lager × ale yeast hybrids, the resistance pattern of all of the hybrids resembled that of the ale yeast parent (Figure 6A, B). Thus, similarly to the initial experiment, the lager × ale hybrids were also more robust and stress‐resistant than lager yeast strains.

Figure 5 Open in figure viewer PowerPoint Generation of lager × ale hybrids via micromanipulation and mating type PCR. (A) Cells of opposite mating type of lager and ale yeast spore clones were mixed and cells resembling zygotes were isolated via micromanipulation. (B) Derived colonies of these zygotes were analysed by mating type PCR. Only strains containing both MATa and MATα bands are hybrids. Here the identification of seven of the 13 lager × ale hybrids is shown. Strains with single bands are, therefore, ale yeast strains. (C) The same colonies as in (B) were assayed for the S. bayanus ORF YBR033W, using specific primers that do not amplify the S. cerevisiae YBR033W homologue

Figure 6 Open in figure viewer PowerPoint Stress profile of lager × ale hybrids compared with the parental strains. The novel lager × ale hybrids and their parental strains were grown and spotted as described in Figure 1 . Stress conditions tested were high temperature (37°C, A) and high salt (B). Pictures were taken after 5 days of growth

The fermentation properties of these lager × ale hybrids were then tested using granulated malt (18°Plato) in 200 ml fermentations at 18°C and 25°C (Figure 7A–D). Most of the strains performed between the ranges of their lager or ale yeast parents. Lager yeast strains could utilize more total sugar of the granulated malt and reached a lower Plato value at the end of fermentation. Most hybrids displayed fermentation performances of the lager type, while fewer performed more similarly to the ale type. As an example, strain C601 showed a typical ale yeast fermentation curve, while another, C612, showed a lager yeast fermentation profile. Interestingly, C612 could consistently utilize more sugars from the granulated malt than its lager yeast parent and also shortened the total fermentation time (Figure 7, Table 2).

Figure 7 Open in figure viewer PowerPoint Analysis of fermentation performance of lager × ale yeast hybrids. Representative fermentation profiles are shown of lager yeast, ale yeast and two hybrids thereof, C601 and C612, which showed most pronounced characteristics. Fermentations were done using high‐gravity granulated malt at 18°C (A, B) and 25°C (C, D). Sugar consumption is indicated by the decrease in °Plato over time (A, C) and weight loss by CO 2 release is indicated in (B, D) (g/l). (E) The amounts of viable cells for each strain were quantified

Table 2. End‐of‐fermentation performance of yeast strains Strain Final °Plato at 18°C Final weight loss (g) at 18°C Final °Plato at 25°C Final weight loss (g) at 25°C Lager yeast 4.23 ± 0.06 10.55 ± 0.26 4.18 ± 0.10 10.37 ± 0.34 Ale yeast 5.00 ± 0.10 10.05 ± 0.23 5.53 ± 0.21 9.52 ± 0.33 C601 5.77 ± 0.15 9.26 ± 0.54 5.80 ± 0.14 9.17 ± 0.36 C612 3.93 ± 0.06 11.06 ± 0.43 3.90 ± 0.08 10.79 ± 0.37

To analyse whether the increased robustness of the lager × ale hybrids also resulted in better survival at the end of fermentation, we quantified the total amount of cells and the viable colony‐forming cells after fermentation at 18°C was completed (Figure 7E). This indicated that the lager × ale hybrids, similar to the ale parental strain, generally had an increased total cell count and also an increase in the fraction of viable cells compared to the lager yeast parental strain. However, we did not identify a strain that was improved in these properties compared to the ale yeast parent.

During hybridization we expected that the hybrids would maintain only one type of mitochondrial DNA (uniparental inheritance). Therefore, we used restriction length polymorphisms of ale and lager COX2 genes to determine the mito‐DNA inheritance in our lager × ale hybrids (Figure 8A). Ten of these strains carried ale mito‐DNA, two lager mito‐DNA and one strain was found to be petite, which corresponds to the colony morphology of this strain and its inability to utilize glycerol as carbon source. Within our set of hybrids, two strains, C604 and C605, were derived from the same parental spore clones. Thus, these strains are isogenic and differ only in their mito‐DNA. The fermentation profiles of both strains, using 18°Plato/18°C as fermentation conditions, were very similar (Figure 8B). Furthermore, our best performing isolate, C612, was also found to contain ale‐type mito‐DNA.