The two parental strains were S. cerevisiae VTT-A81062 (VTT Culture Collection, Finland), a brewer’s yeast strain originally sourced from an ale beer from the United Kingdom, and the S. eubayanus type strain VTT-C12902 (VTT Culture Collection, Finland; deposited as CBS12357 at CBS-KNAW Fungal Biodiversity Centre). The four hybrid strains (A81062 × C12902) that were chosen for further characterization were named H1–H4. Natural auxotrophic mutants (lys- and ura-) of the parental strains were selected on α-aminoadipic and 5-fluoroorotic acid agar plates, respectively [4, 50]. Auxotrophy was confirmed by the inability to grow on minimal selection agar medium (0.67 % Yeast Nitrogen Base without amino acids, 3 % glycerol, 3 % ethanol and 2 % agar).
For the generation of ascospores, the auxotrophic mutants of the parental strains were first grown overnight in YPM medium (1 % yeast extract, 2 % peptone, 4 % maltose) at 20 °C. The yeast was then inoculated into pre-sporulation medium (0.8 % yeast extract, 0.3 % peptone, 10 % glucose) at an OD600 of 0.3 and allowed to grow for 20 h at 20 °C. The yeast was then washed with 1 % potassium acetate and a thick suspension was plated onto sporulation agar (1 % potassium acetate, 10 mg/L lysine and uracil, 2 % agar). The yeast was allowed to sporulate for 7 days at 25 °C. Sporulation efficiency was calculated by counting the frequency of ascospores stained with malachite green . Spore viability was calculated by dissecting ascospores treated with Zymolyase 100T (US Biological, USA) on YPD agar with a micromanipulator .
Generation of interspecific hybrids
Interspecific hybrids between a ura- isolate of S. cerevisiae A81062 and a lys- isolate of S. eubayanus C12902 were produced by first generating ascospores of the auxotrophic mutants as above. Ascospores were scraped off the agar into 1 ml sterile reverse-osmosis purified H2O in 2 ml Eppendorf tubes. Tubes were centrifuged at 5,000×g for 5 min and the supernatant was removed. Ascus walls were digested by the addition of 50 μl 1 mg/ml Zymolyase 100T and incubation at 30 °C for 20 min. 200 μl of sterile H2O was then added, and cells were resuspended by vortexing. 100 μl of the resulting suspensions from both parental strains, with complementary auxotrophic markers, were transferred together to 1 ml YPM medium in a sterile 2 ml Eppendorf tube. Tubes were vortexed and incubated statically at 25 °C for 7 days. After incubation, the tubes were centrifuged at 5,000×g for 5 min and the supernatant was removed. 500 μl of starvation medium (0.1 % yeast extract and 0.1 % glucose) was added, and tubes were incubated for at least 2 h at room temperature. Tubes were then vortexed, after which the approximate cell concentration of the resulting suspension was measured with a NucleoCounter YC-100 (ChemoMetec, Denmark) and 100 μl aliquots were spread onto minimal selection agar (without uracil or lysine). Plates were incubated at 25 °C, and prototrophic colonies (i.e. potential hybrids) appeared after 3–7 days. Colonies were counted and purified by replating on minimal selection agar.
Confirmation of hybrid status by PCR and RFLP
The hybrid status of isolates was confirmed by amplification of rDNA-PCR (ITS1, 5.8S and ITS2) using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and digestion of amplicons using the HaeIII restriction enzyme (New England BioLabs, USA) as described previously . Identification was based on the number of restriction fragments generated by enzyme digestion. S. eubayanus yielded a 3-band pattern (490, 225, 140 bp), S. cerevisiae a 4-band pattern (320, 225, 180, 140 bp), while successful hybrids yielded a pattern with all 5 bands (490, 320, 225, 180 and 140 bp).
Amplification of the S. eubayanus-specific FSY1 gene (amplicon size 228 bp) and the S. cerevisiae-specific MEX67 gene (amplicon size 150 bp) was also performed on the DNA extracted from the hybrid strains using the primers SeubF3 (5′-GTCCCTGTACCAATTTAATATTGCGC-3′), SeubR2 (5′-TTTCACATCTCTTAGTCTTTTCCAGACG-3′), ScerF2 (5′-GCGCTTTACATTCAGATCCCGAG-3′), and ScerR2 (5′-TAAGTTGGTTGTCAGCAAGATTG-3′) as described by Muir et al.  and Pengelly and Wheals . Hybrids were i€dentified by the presence of both genes.
Confirmation of hybrid status by PFGE
Yeast strains were propagated in YPM at 20 °C to an OD600 >1 and then harvested by centrifugation (3,000×g, 5 min, 4 °C). Supernatants were decanted, and cells were resuspended in 10 ml of 4 °C 50 mM EDTA (pH 8). Cell concentrations were determined with a Nucleocounter® YC-100™ (ChemoMetec) and 1.2 × 108 cells were placed in each sample plug. Sample plugs were prepared with the CHEF Genomic DNA Plug Kit for Yeast (Bio-Rad) according to the manufacturer’s instructions.
Sample plugs were loaded into the wells of a 1.0 % pulse field certified agarose (Bio-Rad) gel. PFGE was performed at 14 °C in 0.5× TBE buffer [89 mMTris, 89 mM boric acid, 2 mM EDTA (pH 8)]. A CHEF Mapper XA pulsed field electrophoresis system (Bio-Rad) was used with the following settings: 6 V/cm in a 120° angle, pulse length increasing linearly from 26 to 228 s, and total running time of 38 h. A commercial chromosome marker preparation from S. cerevisiae strain YNN295 (Bio-Rad) was used for molecular mass calibration. After electrophoresis, the gels were stained with ethidium bromide and scanned with Gel Doc XR+ imaging system (Bio-Rad).
DNA content by flow cytometry
Flow cytometry was performed on the yeast strains essentially as described by Haase and Reed . Cells were grown overnight in YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose), and approximately 1 × 107 cells were washed with 1 mL of 50 mM citrate buffer. Cells were then fixed with cold 70 % ethanol and incubated at room temperature for 1 h. Cells were then washed with 50 mM citrate buffer, resuspended in 50 mM citrate buffer containing 0.25 mg mL−1 RNAse A and incubated overnight at 37 °C. 1 mg mL−1 of Proteinase K was then added, and cells were incubated for 1 h at 50 °C. Cells were then stained with SYTOX Green (2 μM; Life Technologies, USA), and their DNA content was determined using a FACSAria cytometer (Becton–Dickinson). DNA contents were estimated by comparing fluorescence intensities with those of S. cerevisiae haploid (CEN.PK113-1A) and diploid (CEN.PK) reference strains. Measurements were performed on duplicate independent yeast cultures, and 100,000 events were collected per sample during flow cytometry.
Characterization of hybrid strains
Four randomly selected hybrids (H1–H4) and the parental strains were chosen for further characterization in a small-scale wort fermentation performed at 12 °C. Yeast was propagated essentially as described previously , with the use of a ‘Generation 0’ fermentation prior to the actual experimental fermentations. The experimental fermentations were carried out in duplicate, in 2-L cylindroconical stainless steel fermenting vessels, containing 1.5 L of wort medium. The wort was produced at the VTT Pilot Brewery from barley malt and wheat malt, and contained an extract content of 12.0 ° Plato (59 g maltose, 19 g maltotriose, 16 g glucose, and 4.6 g fructose per litre) and free amino nitrogen (FAN) content of 269 mg L−1. Yeast was inoculated at a rate of 4 g fresh yeast per litre of wort (corresponding to 16 × 106 viable cells mL−1). The wort was oxygenated to 18 mg L−1 prior to pitching. The fermentations were carried out at 12 °C for 11 days, or until no change in residual extract was observed for 24 h. Wort samples were drawn regularly from the fermentation vessels with a syringe, and placed directly on ice, after which the yeast was separated from the fermenting wort by centrifugation (9,000×g, 10 min, 1 °C).
Flocculation of the yeast strains was evaluated using a modified Helm’s assay essentially as described by D’Hautcourt and Smart . Cultures recovered from fermentation were washed twice with 0.5 M EDTA (pH 7) to break the cell aggregates and then diluted to an OD600 of 0.4. Flocculation was assayed by first washing yeast pellets with 4 mM CaCl2 solution and resuspending them in 1 ml of flocculation solution containing 4 mM CaCl2, 6.8 g/L sodium acetate, 4.05 g/L acetic acid, and 4 % (v/v) ethanol (pH 4.5). Yeast cells in control tubes were resuspended in 0.5 M EDTA (pH 7). After a sedimentation period of 10 min, samples (200 μL) were taken from just below the meniscus and dispersed in 10 mM EDTA (800 μL). The absorbance at 600 nm was measured, and percentage of flocculation was determined from the difference in absorbance between control and flocculation tubes.
Maltose and maltotriose uptake by the yeast strains was assayed by first growing them in YPM medium at 20 °C. Yeasts were usually harvested at an OD600 nm between 4 and 8 (i.e. at 2 ± 1 mg dry yeast mL−1) by centrifugation, washed with ice–cold water and then with ice–cold 0.1 M tartrate-Tris (pH 4.2) and finally resuspended in the same buffer at a concentration of 200 mg of fresh yeast mL−1. Zero-trans rates of [U-14C]-maltose and [U-14C]-maltotriose uptake at 20 °C were determined with 5 mM substrate in 0.1 M tartrate-Tris (pH 4.2) as described earlier , with reaction time of 1 min. [U-14C]-maltose (ARC 488) and [U-14C]-maltotriose (ARC 627) were from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). [U-14C]-maltotriose was repurified before use as described by Dietvorst et al. .
Wort and beer analysis
The specific gravity, alcohol level and pH of samples were determined from the centrifuged and degassed fermentation samples using an Anton Paar density meter DMA 5000 M (Anton Paar GmbH, Austria) with Alcolyzer Beer ME and pH ME modules (Anton Paar GmbH, Austria).
The yeast dry mass content of the samples (i.e. yeast in suspension) was determined by washing the yeast pellets gained from centrifugation twice with 25 mL deionized H2O and then suspending the washed yeast in a total of 6 mL deionized H2O. The suspension was then transferred to a pre-weighed porcelain crucible and was dried overnight at 105 °C and allowed to cool in a desiccator, before the change of mass was measured.
Concentrations of fermentable sugars (glucose, fructose, maltose and maltotriose) were measured by HPLC using a waters 2695 separation module and waters system interphase module liquid chromatograph coupled with a waters 2414 differential refractometer (Waters Co., Milford, MA, USA). An Aminex HPX-87H organic acid analysis column (300 × 7.8 mm, Bio-Rad) was equilibrated with 5 mM H2SO4 (Titrisol, Merck, Germany) in water at 55 °C and samples were eluted with 5 mM H2SO4 in water at a 0.3 ml/min flow rate.
Yeast-derived flavour compounds were determined by headspace gas chromatography with flame ionization detector (HS-GC-FID) analysis. 4 mL of samples was filtered (0.45 µm), incubated at 60 °C for 30 min and then 1 mL of gas phase was injected (split mode; 225 °C; split flow of 30 mL min−1) into a gas chromatograph equipped with a FID detector and headspace autosampler (Agilent 7890 Series; Palo Alto, CA, USA). Analytes were separated on a HP-5 capillary column (50 m × 320 µm × 1.05 µm column, Agilent, USA). The carrier gas was helium (constant flow of 1.4 mL min−1). The temperature program used 50 °C for 3 min, 10 °C min−1 to 100 °C, 5 °C min–1 to 140 °C, 15 °C min−1 to 260 °C and then isothermal for 1 min. Compounds were identified by comparison with authentic standards and were quantified using standard curves. 1-Butanol was used as internal standard.