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Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Elena S Naumova, Irina V Korshunova, Lene Jespersen, Gennadi I Naumov
DOI: http://dx.doi.org/10.1016/S1567-1356(02)00191-5 177-184 First published online: 1 April 2003

Abstract

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)5 and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.

Keywords
  • Sorghum beer
  • UP-PCR
  • ITS1 sequencing
  • Southern hybridisation
  • SUC and RTM genes
  • Saccharomyces sensu stricto

1 Introduction

Spontaneously fermented foods and beverages contribute significantly to the indigenous diet in several countries in West Africa. Sorghum beer is a traditional alcoholic beverage of this region [14]. Identification studies on yeasts isolated from sorghum beer have revealed that the yeast species composition varied with different production sites. Saccharomyces cerevisiae was found to predominate in Ghanaian sorghum beer, making up 33–38% of the yeast cell population [1,5]. In sorghum beer from Togo and Burkina Faso, Saccharomyces yeasts made up 55–90% of the cell population [2]. In a previous study [6], we have examined the yeast population of sorghum beer in the northern part of Ghana and Burkina Faso and found that it consists almost exclusively of Saccharomyces sensu stricto strains. The Saccharomyces strains involved in sorghum beer fermentation were very heterogeneous for phenotypic properties. Of 100 isolates representing four production sites, half were limited to assimilation of only glucose and maltose, whereas the other half displayed a broader assimilation spectrum more typical of S. cerevisiae. Restriction fragment length polymorphism (RFLP) analysis of the ribosomal internal transcribed spacer region, molecular karyotyping and sequencing of the D1/D2 domain of the 26S rDNA suggested close relatedness of all isolates to S. cerevisiae, regardless of their phenotypic properties.

Within the Saccharomyces sensu stricto complex, six sibling species have been established: S. cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae and S. paradoxus[7,8]. The seventh sterile taxon S. pastorianus (syn. S. carlsbergensis Hansen) is not a biological species but a hybrid probably originated from S. cerevisiae×S. bayanus[911]. The six sibling species have a common mating-type system allowing them to cross in any combination. The resulting hybrids, however, are sterile, yielding non-viable ascospores. In contrast, intraspecific hybrids are fertile and have regular segregation of control markers [8,12]. The six sibling species possess common basic karyotypic characters: the same range of chromosomal bands (from 245 to 2200 kb) and the same haploid number of chromosomes (n=16). Two species, S. bayanus and S. cariocanus, have species-specific karyotypes, while the other members of the Saccharomyces sensu stricto complex cannot be distinguished on the basis of their banding patterns [12].

The accumulation of multiple β-fructosidase SUC genes controlling sucrose fermentation has been observed in some industrial populations of S. cerevisiae[13,14]. In particular, distiller's and baker's strains, which are industrially cultivated on molasses containing sucrose, carry several SUC genes, whereas wine yeasts, which are not cultivated on sucrose, usually show a single SUC2 gene. It is known that grape juice contains fructose and glucose, but not sucrose. Polymeric SUC genes have been mapped in the telomeric regions of different chromosomes: SUC1 on chromosome VII, SUC3 on II, SUC4 on XIII, SUC5 on IV, and SUC7 on VIII [15,16]. One exception is the SUC2 gene, which is located at the end of chromosome IX but not in the telomeric region. Recently, a new family of telomeric RTM genes (leading to resistance to toxic molasses) has been described [14]. In distiller's and baker's yeasts, the RTM genes are always physically associated with the SUC telomeric loci. Only the non-telomeric SUC2 gene is not associated with the RTM sequence. Although brewing yeasts are grown mainly on maltose as a carbon source, both top- and bottom-fermenting strains have been shown to contain many telomeric SUC genes and RTM sequences [17]. The presence of multiple telomeric SUC genes may indicate the common genetic origin of European brewing, baker's and distiller's yeasts.

In the present work, we compare 24 phenotypically different strains isolated from sorghum beer in Ghana and Burkina Faso with the type cultures of the six Saccharomyces sensu stricto species and the hybrid taxon S. pastorianus. Our purpose is to elucidate the taxonomic position of African sorghum beer strains and to estimate genetic diversity among the isolates. We also examine the discriminatory power of RFLP analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal gene for separation of seven members within the Saccharomyces sensu stricto complex.

2 Materials and methods

2.1 Yeast strains

Samples of dry yeasts were collected from two commercial production sites in northern Ghana (A and B) and two sites in Burkina Faso (C and D) [6]. The 24 phenotypically different strains used in this study and their physiological characteristics are listed in Table 1. Yeasts were grown at 28°C on complete YPD medium (1% yeast extract, 2% peptone, 2% glucose and 2% agar). Sporulation was obtained on acetate medium (1% CH3COONa, 0.5% KCl and 2% agar). Spores were isolated from asci by a micromanipulator. The type cultures of the Saccharomyces sensu stricto species were used in molecular experiments: S. cerevisiae CBS 1171, S. bayanus CBS 380, S. cariocanus NCYC 2890 (=CBS 8841), S. kudriavzevii NCYC 2889 (=CBS 8840), S. mikatae NCYC 2888 (=CBS 8839), S. paradoxus CBS 432 and S. pastorianus CBS 1538.

View this table:
Table 1

African Saccharomyces sensu stricto strains used and their physiological properties

StrainsaAssimilation profilesb
A6, A15, A19, A22, B5, B16, B20, B24, C4, C6, C11, C19, D3, D11Suc Mal+ Galα-Mgl Mlz Pal Raf Tre
B7, D12, D21Suc+ Mal+ Galα-Mgl Mlz Pal Raf Tre
A7Suc+ Mal+ Galα-Mgl Mlz+ Pal+ Raf Tre
C24Suc+ Mal+ Galα-Mgl+ Mlz Pal+ Raf Tre
C10Suc+ Mal+ Galα-Mgl+ Mlz+ Pal+ Raf Tre
A10Suc+ Mal+ Galα-Mgl+ Mlz+ Pal+ Raf Tre+
D1Suc+ Mal+ Galα-Mgl Mlz Pal Raf+ Tre
C1Suc+ Mal+ Gal+α-Mgl Mlz+ Pal+ Raf Tre+
A12Suc+ Mal+ Gal+α-Mgl+ Mlz+ Pal+ Raf+ Tre+
  • aA, B and C, D are different production sites in northern Ghana and Burkina Faso, respectively.

  • bPhenotypic characters are given according to van der Aa Kühle et al. [6]. Suc, sucrose; Mal, maltose; Gal, galactose; α-Mgl, α-methyl-d-glucoside; Mlz, melizitose; Pal, palatinose; Raf, raffinose; Tre, trehalose.

2.2 RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rDNA region; ITS1 sequence analysis

Isolation of DNA was performed as previously described [18]. The primers used for amplification of the ribosomal DNA containing the internal transcribed spacer 1, 5.8S rDNA, and internal transcribed spacer 2 were ITS1 (5′-TCCGTAGGTGAACCTGCGG) and ITS4 (5′-TCCTCCGCTTATTGATATGC) as described by White et al. [19]. Amplification reactions were performed in a volume of 50 μl containing 100 ng of genomic DNA, 0.2 mM of each dNTP, 50 pM of each primer, 5 μl 10×PCR buffer (Amersham Pharmacia Biotech, Sunnyvale, CA, USA) and 2.5 U Taq polymerase (Amersham Pharmacia Biotech). The thermal cycler was programmed for 30 cycles of 2 min at 94°C, 1 min at 60°C and 1 min at 72°C. Amplification products were separated by electrophoresis in 1.2% agarose gels and detected by staining with ethidium bromide.

For RFLP analysis, 10 μl PCR products were digested with 5 U of each of the following five restriction enzymes: HaeIII, HinfI, HpaII, ScrFI and TaqI (New England BioLabs, Beverly, MA, USA) overnight at 37°C. The resulting fragments were separated on 2.5% agarose gels in 1×TAE buffer. The restriction fragments were visualised by ethidium bromide staining under UV light. Lambda DNA/HindIII and 174/HaeIII markers (Promega, Madison, WI, USA) were used as the size standards.

For sequencing of the ITS1 region, the amplified products were purified using the Qiagen PCR purification kit (Qiagen, Dorking, UK). The purified PCR products were directly sequenced by using a Thermo Sequenase fluorescent-labelled primer cycle sequencing kit (RPN 2436, Amersham Pharmacia Biotech), following the manufacturer's instructions, in an automated DNA sequencer (ALFexpress, Amersham Pharmacia Biotech). To sequence both strands, two 5′-Cy5-labelled primers ITS1 (as described above) and ITS2 (5′-GCTGCCCCCCGTTCTTCATCGATGC) were used. Sequence data were visually aligned with ClustalW (EMBL-European Bioinformatics Institute, Cambridge, UK). The nucleotide sequences determined for strains A12 (=CBS 8856) and D3 (=CBS 8858) have been deposited with GenBank under the following accession numbers: AY115573 and AY115574, respectively.

2.3 UP-PCR analysis and microsatellite fingerprinting

UP-PCR (universally primed PCR) [20] is an analogue of RAPD-PCR (randomly amplified polymorphic DNA PCR) [21] based on the amplification of genomic DNA with single short primers of random nucleotide sequence. The main difference consists of the special design of primers (longer primers containing the core part and the variable 3′ end) and PCR conditions (relatively high annealing temperatures). The advantage of UP-PCR over RAPD is its reproducibility, which many have found to be problematic with RAPDs [20]. The PCR was performed in 30 μl containing 100 ng of genomic DNA, 0.6 mM of each dNTP, 50 pM of a primer, 10×PCR buffer (20 mM Tris–HCl, pH 8.8, 10 mM (NH4)2SO4, 4 mM MgCl2) and 2 U DynaZyme version 2.0 polymerase (Finnzymes, Espoo, Finland). The sequence of the universal primer N21 is 5′-GGATCCGAGGGTGGCGGTTCT.

The thermal cycler was programmed for 30 cycles of 50 s at 94°C, 80 s at 55°C and 60 s at 72°C. Amplification products were separated by electrophoresis in 1.2% agarose gels and detected by staining with ethidium bromide.

Total UP-PCR products generated from each strain with the primer N21 were spotted onto nitrocellulose filters (Hybond C, Amersham, UK). The probes for hybridisations were labelled with digoxigenin-11-dUTP using the DIG High Prime DNA Labelling and Detection Starter Kit I (Boehringer Mannheim, Mannheim, Germany). Hybridisation and colorimetric detection were performed as recommended by the supplier.

For microsatellite fingerprinting with primer (GTG)5, amplification reactions were performed in a volume of 30 μl containing 25–100 ng of genomic DNA, 0.2 mM of each dNTP, 50 pM primer, 3 μl 10×PCR buffer (Amersham Pharmacia Biotech) and 2.5 U Taq polymerase (Amersham Pharmacia Biotech). The thermal cycler was programmed for 30 cycles of 30 s at 94°C, 30 s at 52°C and 60 s at 72°C. Amplification products were separated by electrophoresis in 1.2% agarose gels and detected by staining with ethidium bromide.

2.4 Southern hybridisation analysis

Preparation of chromosomal DNAs has been described elsewhere [22]. A CHEF-DR II apparatus (Bio-Rad, Hercules, CA, USA) was used to separate chromosomal DNA. The electrophoresis buffer (0.5×TBE) was circulated around the gel and cooled to 14°C. Electrophoresis was conducted at 200 V for 15 h with a switching time of 60 s followed by 9 h with a switching time of 90 s. S. cerevisiae strain YNN 295 (=ATCC 200358) was used as a karyotype standard (Bio-Rad). The chromosomal DNA separated by CHEF was Southern- blotted onto nitrocellulose filters. The SUC2 probe was a 0.9-kb BamHI–HindIII fragment isolated from pRB117 [15]. The RTM1 probe was a 0.9-kb EcoRI–BamHI fragment isolated from p1K [14]. The probes were labelled with digoxigenin-11-dUTP using the DIG High Prime DNA Labelling and Detection Starter Kit I (Boehringer Mannheim, Mannheim, Germany). Hybridisation and colorimetric detection were performed as recommended by the supplier.

3 Results

3.1 Genetic identification of strains

The 24 phenotypically different strains isolated from sorghum beer (Table 1) were first tested for sexual capacity. 20 of them (A7, A10, A15, A19, B5, B7, B16, B24, C1, C4, C6, C10, C11, C19, C24, D1, D3, D11, D12 and D21) did not sporulate at all. Four sporulating isolates were subjected to tetrad analysis. Three isolates (A6, A12 and A22) showed very low ascospore viability and one strain, B20, had non-viable ascospores. Due to self-sterility or very low fertility, the genetic identification of these strains was not possible. The genetic relatedness of the 24 strains was further analysed using molecular methods.

3.2 PCR-RFLP analysis of the ITS1–5.8S–ITS2 (ITS) region of rDNA

The ITS regions from the type cultures of S. cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus, S. pastorianus and from the 24 African sorghum beer isolates were amplified using primers ITS1 and ITS4. The ITS region of all strains studied showed the same molecular size of about 850 bp. The PCR products were digested with five restriction endonucleases: HaeIII, HinfI, HpaII, ScrFI and TaqI (Table 2). The RFLP analysis did not allow the separation of S. bayanus from S. pastorianus and of S. cariocanus from S. paradoxus. However, S. cerevisiae, S. kudriavzevii and S. mikatae could be clearly differentiated from each other and from the pairs mentioned above by using a combination of the endonucleases HaeIII, HpaII, ScrFI and TaqI. The latter restriction enzyme only discriminated between S. kudriavzevii and the remaining members of the Saccharomyces sensu stricto complex.

View this table:
Table 2

Restriction analysis of the ITS1–5.8S–ITS2 region from Saccharomyces sensu stricto strains

Species designationStrainRestriction fragments
HaeIIIHinfIHpaIIScrF1TaqI
S. cerevisiaeCBS 1171 (T)a325+230+170+125375+365+110725+125400+320+130290+250+130+110+70
S. bayanusCBS 380 (T)495+230+125375+365+110725+125400+320+130290+250+130+110+70
S. cariocanusNCYC 2890 (T)325+230+170+125375+365+110850530+320290+250+130+110+70
S. kudriavzeviiNCYC 2889 (T)495+230+125375+365+110725+125400+320+130320+290+130+110
S. mikataeNCYC 2888 (T)495+230+125375+365+110850530+320290+250+130+110+70
S. paradoxusCBS 432 (T)325+230+170+125375+365+110850530+320290+250+130+110+70
S. pastorianusCBS 1538 (T)495+230+125375+365+110725+125400+320+130290+250+130+110+70
SaccharomycesSorghum beer yeastsb325+230+170+125375+365+110725+125400+320+130290+250+130+110+70
  • aT, type culture.

  • bStrains analysed: A6, A7, A10, A12, A15, A19, A22, B5, B7, B16, B20, B24, C1, C4, C6, C10, C11, C19, C24, D1, D3, D11, D12 and D21.

All 24 sorghum beer strains shared the same restriction patterns with endonucleases HaeIII, HpaII, ScrFI and TaqI, and these patterns were identical to those obtained for the S. cerevisiae type culture (Table 2). This suggests that all strains isolated from African sorghum beer belong to S. cerevisiae.

3.3 PCR analysis and dot blot hybridisation

PCR profiles generated with universal primer N21 were nearly identical for all 24 isolates and for the type culture of S. cerevisiae (Fig. 1, lane 1). No differences were found between strains with narrow and broad assimilation spectra (Table 1). However, the type culture of S. bayanus displayed a clearly different profile with many minor bands (Fig. 1, lane 6). The UP-PCR products of the type cultures of S. cerevisiae and S. bayanus were used as labelled probes in dot blot hybridisations. Strong hybridisation signals were obtained for all sorghum beer strains only with the S. cerevisiae probe (Fig. 2A). In contrast, the S. bayanus probe showed hybridisation only to its own DNA (Fig. 2B).

Figure 1

UP-PCR banding profiles for sorghum beer strains and the type cultures of S. cerevisiae (CBS 1171) and S. bayanus (CBS 380), generated with primer N21. Lanes: 1, CBS 1171; 2, A6; 3, A20; 4, B5; 5, C1; 6, D1; 7, CBS 380. M, molecular mass marker Lambda DNA/HindIII (Promega) is indicated in base pairs.

Figure 2

Dot blot hybridisation of UP-PCR products generated with the primer N21 for sorghum beer strains. The PCR products were spotted onto nitrocellulose filters and hybridised with the labelled total UP-PCR product of the type cultures of S. cerevisiae CBS 1171 (A) and S. bayanus CBS 380 (B). S. cerevisiae: 1, CBS 1171; 2, A6; 3, A12; 4, A20; 5, A22; 6, B5; 7, B12; 8, B20; 9, B22; 10, C1; 11, C4; 12, C6; 13, D1; 14, D12; 15, D21. S. bayanus: 16, CBS 380.

PCR fingerprinting with the microsatellite primer (GTG)5 also did not reveal significant heterogeneity among sorghum beer yeasts; the 24 strains exhibited very similar banding patterns (not shown).

3.4 Sequence analysis of phenotypically different strains

Two sorghum beer strains having different assimilation profiles were further compared by sequence analysis of the ITS1 region. Strain D3 isolated in Burkina Faso can assimilate only glucose and maltose, whereas strain A12 from Ghana can assimilate many sugars (Table 1). The two strains showed identical sequences with 0.5% nucleotide difference from the type culture of S. cerevisiae. Using the BLAST programme we compared the ITS1 sequence of the strains isolated from sorghum beer with ITS1 sequences available in the database for other S. cerevisiae strains (Fig. 3). The ITS1 sequence of sorghum beer strains differs from all other strains, including the type culture CBS 1171, by a three-nucleotide insertion (ACA) located at the position 213–215 (numbering based on the ITS1 sequence of A12/D3). The main difference among the ITS1 sequences of the other S. cerevisiae strains is the number of thymines (from seven to 14) at the position 28–39 of the ITS1 of A12/D3. This apparent variability is probably a consequence of sequence errors. For example, there are two nucleotide sequences of the ITS1 region of the type culture of S. cerevisiae CBS 1171 (=IFO 10217) (accession numbers AB018043 and D89886) obtained by different authors that differ in number of thymines at this position. The three-nucleotide insertion observed in sorghum beer strains is also absent in the ITS1 regions of S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. pastorianus.

Figure 3

Alignment of the ITS1 sequences of the sorghum beer strains (A12 and D3) and the type culture of S. cerevisiae (CBS 1171=IFO 10217). Underlined nucleotides represent the insertion in the ITS1 region of A12/D3.

3.5 Molecular karyotyping and Southern hybridisation analysis

All 24 isolates showed karyotype patterns typical of S. cerevisiae (Fig. 4). Some chromosome length polymorphism was evident in middle-size bands. No correlation was found between karyotype patterns and assimilation spectra of the strains.

Figure 4

Chromosomal patterns of S. cerevisiae strains isolated from sorghum beer in northern Ghana and Burkina Faso. Lanes: 1, YNN 295 (standard); 2, CBS 403; 3, A6; 4, A7; 5, A10; 6, A12; 7, A15; 8, A19; 9, A22; 10, B5; 11, B7; 12, B16; 13, B20; 14, B24; 15, C1; 16, C4; 17, C6; 18, C10; 19, C11; 20, C19; 21, C24; 22, D1; 23, D3; 24, D21; 25, D11; 26, D12. The chromosome sizes refer to the chromosomes of the standard strain YNN 295.

We tested for the presence of SUC and RTM genes in 24 strains isolated from sorghum beer in Ghana and Burkina Faso. Both sucrose-positive and sucrose-negative strains were found to carry only a single SUC2 gene (Fig. 5, lanes 5–28) and no RTM genes (not shown). For comparison, strain CBS 403, which was isolated from ginger beer in West Africa at the beginning of the last century, has SUC2 and two telomeric genes, SUC1 and SUC3, associated with the RTM sequences (Fig. 5, lane 4). The type culture of S. cerevisiae CBS 1171, which was isolated from top beer fermentation in Europe, showed seven SUC loci: SUC1, SUC2, SUC3 and four apparently new SUC genes indicated by hybridisation with the SUC2 probe at new locations (chromosomes XIV, XI, III and VI) (Fig. 5, lane 2). The six SUC genes of strain CBS 1171 were associated with RTM sequences (not shown).

Figure 5

Southern blot analysis of the chromosomal locations of SUC genes in African sorghum beer strains. Lanes: 1 and 3, YNN 295 (standard); 2, CBS 1171; 4, CBS 403; 5, A6; 6, A7; 7, A10; 8, A12; 9, A15; 10, A19; 11, A22; 12, B5; 13, B7; 14, B16; 15, B20; 16, B24; 17, C1; 18, C4; 19, C6; 20, C10; 21, C11; 22, C19; 23, C24; 24, D1; 25, D3; 26, D21; 27, D11; 28, D12. The linkage group numbering refers to the chromosomes of the standard strain YNN 295.

4 Discussion

Restriction analysis of the rDNA region spanning the 5.8S rRNA gene and flanking internal transcribed spacers (ITS1 and ITS2) has been shown to be a rapid and easy method for the identification of various yeasts [2325]. Recently, Fernández-Espinar et al. [26] have proposed the use of the ITS-PCR RFLP with endonucleases ScrFI and HpaII for differentiation of S. paradoxus from S. cerevisiae and from the two indistinguishable yeasts S. bayanus and S. pastorianus. In the present study, we have extended RFLP analysis of the entire ITS region to the newly described species S. cariocanus, S. kudriavzevii and S. mikatae. Our results indicate that only a combination of the endonucleases HaeIII, HpaII, ScrF1, and TaqI enables the discrimination of S. cerevisiae, S. kudriavzevii and S. mikatae from one another and from the S. cariocanus/S. paradoxus and S. bayanus/S. pastorianus pairs. S. cariocanus cannot be distinguished from S. paradoxus and S. bayanus nor from S. pastorianus on the basis of the restriction patterns obtained with any of the four enzymes or their combinations. This result is in good agreement with the sequencing data on the ITS1 region, showing that S. cariocanus is most closely related to S. paradoxus and S. pastorianus to S. bayanus[8,27].

With the five endonucleases assayed, the 24 sorghum beer isolates showed restriction patterns identical to those of the S. cerevisiae type culture. Therefore, despite their phenotypic variability, all strains belong to S. cerevisiae. UP-PCR analysis gave concordant results. PCR fingerprinting with the microsatellite primer (GTG)5 was shown to be a reliable tool for molecular typing of individual strains of S. cerevisiae and S. paradoxus[28,29]. However, this primer cannot be used in distinguishing sorghum beer isolates due to their nearly identical (GTG)5 profiles. Surprisingly, the phenotypically different sorghum beer strains did not show a high degree of variation at the molecular level. Cultivated S. cerevisiae strains usually have a great deal of chromosome length polymorphism [30,31]. In contrast, African sorghum beer strains exhibited a much lower variation both in number and size of chromosomal bands as compared to other brewer's strains [3234].

Earlier, the ITS1 and ITS2 sequences have been used by us to analyse the relatedness of sibling species within the Saccharomyces sensu stricto complex [8]. More detailed resolution was achieved by using the ITS1 region, which showed far greater sequence variation than the ITS2 region. In the present study, we compared the ITS1 regions of two phenotypically different sorghum beer strains. Based on the ITS1 and D1/D2 sequence data (this study, [6]), sorghum beer strains represent a divergent population of S. cerevisiae. Within the population, strains showed identical ITS1 and D1/D2 sequences, with 0.5% nucleotide difference from the type culture of S. cerevisiae. The divergence of the S. cerevisiae sorghum beer population from other S. cerevisiae populations was further confirmed by Southern hybridisation analysis. Unlike European brewing yeasts from top and bottom fermentations, having multiple telomeric SUC and RTM genes, African sorghum beer yeasts carry a single SUC2 gene and no RTM sequences. This suggests a different genetic origin of African S. cerevisiae isolates and European brewing yeasts of S. cerevisiae.

Acknowledgements

The authors would like to thank M. Jakobsen (Copenhagen) for his interest in the work, P. Sniegowski (Philadelphia, PA) for critical reading of the manuscript and the Organisation Committee of ISSY22 for an invitation to present an oral communication. The study was supported by Danida (Danish International Development Assistance) and by a grant from the Russian Fund for Basic Research (N00-04-49116).

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View Abstract