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Correspondence: Alan E. Wheals, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. Tel.: +44 1225 383583; fax: +44 1225 386779; e-mail: email@example.com
The Saccharomyces species Saccharomyces eubayanus was recently discovered in Patagonia. Genomic, genetic and phylogenetic data all suggest that it is one of the two parents of the hybrid yeast S. pastorianus (S. cerevisiae being the other). Saccharomyces eubayanus genomes can also be found in strains of the hybrid species S. bayanus. Here, we describe a novel pair of PCR primers targeted against the S. eubayanus FSY1 gene that will specifically detect S. eubayanus and hybrids containing this allele of the gene. The primer pair can be used to identify the species using a rapid, inexpensive colony PCR method suitable for a preliminary identification of wild isolates.
The natural habitat of members of the monophyletic genus Saccharomyces is in association with trees, bark and fruits. Some species have been domesticated and subjected to strong selection and hybridisation events. Until recently, the species were S. arboricola, S. bayanus var. bayanus, S. bayanus var. uvarum, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus and S. pastorianus. Population genomics revealed that S. cariocanus was an American variety within the S. paradoxus clade (Liti et al., 2009). The exact relationships of S. pastorianus, S. bayanus var. bayanus and S. bayanus var. uvarum have been problematic (Nguyen & Gaillardin, 2005) but have been clarified by the important discovery of the new species S. eubayanus (Libkind et al., 2011) that has also been shown to be one of the two parents of the hybrid S. pastorianus (Nakao et al., 2009). Saccharomyces bayanus var. bayanus CBS 380T also contains significant amounts of S. eubayanus-derived sequences (Libkind et al., 2011). The genus can now be better resolved into seven single-genome species and two hybrid species (S. bayanus and S. pastorianus) with CBS 395T being effectively reinstated as the type strain of S. uvarum (discussed in Libkind et al., 2011). This nomenclature is used henceforth in this article.
We have previously developed primers for the successful specific detection, in single or multiplex format, by PCR amplification at 60 °C of six of the single-genome species (Muir et al., 2011; see also Torriani et al., 2004). Saccharomyces bayanus was then thought to be related to one of the parents of S. pastorianus, so primers designed to detect S. bayanus were used to test the hybrid nature of S. pastorianus. In many cases, the test failed and the likely explanation is now clear; S. cerevisiae is one parent and S. eubayanus is the other. Saccharomyces bayanus may be a double hybrid of S. uvarum × S. pastorianus, thus containing genetic material from three donor species via multiple hybridisation events (Libkind et al., 2011; Nguyen et al., 2011). Our primers previously described as specific for S. bayanus were originally developed on the monogenomic strain CBS 7001. This isolate is clearly S. uvarum (Libkind et al., 2011), and so these primers have been renamed as S. uvarum specific. We report here the successful development of S. eubayanus species-specific primers that also detect isolates or hybrids containing the S. eubayanus allele.
Original designation: *designated S. uvarum based upon Libkind et al. (2011). †Contains a horizontally transferred FSY1 gene (Galeote et al., 2010).
Strain: CBS; Centraalbureau voor Schimmelcultures, Utrecht, Netherlands. IFO is now NBRC (NITE Biological Resource Center), Chiba, Japan. NCYC; National Collection of Yeast Cultures, Norwich, UK. NRRL; Agricultural Research Service, Peoria, USA. SGRP (Liti et al., 2009). UniL; UniLever plc., Colworth, UK. VKM; All-Russian Collection of Microorganisms, Pushchino, Russia. ZP; Gonçalves P. et al. (2011).
Designation ‘Presumptive S. uvarum’ is based on a negative response of a S. bayanus strain to Seub-specific primers in this paper and/or other data.
S. bayanus (S. uvarum/S. eubayanus hybrid); Libkind et al. (2011) and this paper
Both previously published (Muir et al., 2011) and new species-specific primers used are shown with full details (Table 2). The S. uvarum-specific primers were originally designated S. bayanus-specific primers (discussed in Libkind et al., 2011).
Table 2. Species-specific primers (further described in Muir et al., 2011)
Primer sequence (5′ to 3′)
Amplicon size (bp)
GGC ACG CCC TTA CAG CAG CAA
TCG TCG TAC AGA TGC TGG TAG GGC
GCT GAC TGC TGC TGC TGC CCC CG
TGT TAT GAG TAC TTG GTT TGT CG
GCG CTT TAC ATT CAG ATC CCG AG
TAA GTT GGT TGT CAG CAA GAT TG
GTC CCT GTA CCA ATT TAA TAT TGC GC
TTT CAC ATC TCT TAG TCT TTT CCA GAC G
ATC TAT AAC AAA CCG CCA AGG GAG
CGT AAC CTA CCT ATA TGA GGG CCT
ACA AGC AAT TGA TTT GAG GAA AAG
CCA GTC TTC TTT GTC AAC GTT G
CTT TCT ACC CCT TCT CCA TGT TGG
CAA TTT CAG GGC GTT GTC CAA CAG
Sequences and bioinformatics
Gene sequences of S. cerevisiae, S. uvarum, S. kudriavzevii, S. paradoxus and S. mikatae were obtained from www.SaccharomycesSensuStricto.org (Scannell et al., 2011), S. eubayanus was obtained from Libkind et al. (2011), S. arboricola sequences were provided by G. Liti (pers. commun.), and FSY1 sequences are from EBI (www.ebi.ac.uk).
DNA sequence analyses were performed using mega 5.05 (Tamura et al., 2011). Alignments were performed on protein sequences using clustalw utilising the BLOSUM62 substitution matrix and edited manually where required.
Cell and molecular methods
Growth of cells, colony PCR protocols and gel electrophoresis were as described in the study by Muir et al. (2011). As previously described (Muir et al., 2011), primer development and specificity testing was initially performed at 55 °C for use at 60 °C (with no significant differences in the results). A 100 bp DNA size ladder was used in all gels.
Gene selection and primer design
Existing Saccharomyces species-specific primers were tested on S. eubayanus. An example is shown (Fig. 1) where no amplicons were produced in either single or multiplex format with the S. eubayanus type strain but S. bayanusT contains S. uvarum-specific sequences. A small number of gene sequences were available for S. eubayanus primer design and FSY1, a fructose/H+ symporter (Gonçalves et al., 2000), was selected because it is present in S. uvarum (Supporting information, Fig. S1) but absent in other monogenomic Saccharomyces isolates. blast analysis revealed similarity to related symporters in other yeast genera (79% nucleotide identity to the S. eubayanus allele in Zygosaccharomyces rouxii CBS 732), but there is a clear difference between those sequences and sequences derived from S. eubayanus and S. uvarum lineages. A total of eight primers were designed against regions of variability between S. eubayanus and S. uvarum. Amplicon sizes of all possible pairwise primer combinations were calculated and validated experimentally against S. eubayanus; no primer pairs produced an amplicon with S. uvarumT (data not shown). Primer pair Seub F3/R2 with an amplicon size of 228 bp (that would differentiate it from existing primer products; Table 2) was selected for further testing. Temperature gradient PCR showed optimum amplification at the required 60 °C. In a seven-species multiplex format, it worked well with all other existing primers (Fig. S2).
Primer pair Seub F3/R2 was tested against single-genome Saccharomyces species resulting in no positive reactions with type strains of nontarget species (Fig. 2). Seub F3/R2 was tested against species representing the family Saccharomycetaceae, several of which contain orthologues of the target gene, but there were no positive reactions against nontarget species (Fig. S3). Seub F3/R2 was tested against species often isolated from wine fermentations including several known to possess orthologous symporter genes. All nontarget species were negative in the test (Fig. S4).
Both S. pastorianus and S. bayanus isolates contain S. eubayanus sequences that may include the target FSY1 gene. All five isolates of S. pastorianus that were tested were positive (Fig. 3) from which we infer that S. eubayanus DNA is present. Tests of S. bayanus strains in our collection were sometimes positive (confirmed in Fig. S5), but most were not (Fig. 4). These data are consistent with the hypothesis that strains labelled as S. bayanus with a varietal name of either var. bayanus or var. uvarum are in fact two distinct species. Four other hybrids or strains with other kinds of introgressions were also tested, all with negative results (Fig. S6).
Primer pair Seub F3/R2 has the required sensitivity and specificity for detecting the presence of the diagnostic S. eubayanus FSY1 allele. Saccharomyces eubayanus has been genome sequenced but not yet fully annotated. FSY1 (Libkind et al., 2011) was of particular interest because it is present only in the Saccharomyces species S. eubayanus and S. uvarum, in hybrid strains containing these genomes (S. bayanus and S. pastorianus) and in some oenological strains of S. cerevisiae (Galeote et al., 2010; Libkind et al., 2011). Only two homologous sequences from S. eubayanus and S. uvarum were considered during primer design, and several target sequences with at least four mismatches in each primer were found. The absence of FSY1 orthologues in most Saccharomyces species shows that the gene is not essential for cell viability. This may increase the risk of false negatives in the target species due to potential gene loss. A test of this would be informative once there is more than one publicly available isolate.
Given that the majority of the strains designated as S. bayanus in the literature are actually S. uvarum, most were not expected to have the S. eubayanus-derived sequences present (Nguyen & Gaillardin, 2005; Nguyen et al., 2011), and this was confirmed. Of the previously designated S. bayanus and S. uvarum species tested, only five were positive (Fig. 4) in agreement with the data of the study by Libkind et al. (2011; Fig. S2).
The S. pastorianus genome has been shown to be partially derived from S. eubayanus, and this was confirmed in these tests. None of the three hybrids (CID1, CECT 1885 and SPG 16-91) described in the study by Gonzalez et al. (2006) showed positive reactions with the Seub F3/R2 primer pair. Gain of genes by horizontal gene transfer is thought to be rare (Scannell et al., 2007) but may occur more frequently under selection in the brewing and wine industries (Galeote et al., 2010; Scannell et al., 2011). The S. cerevisiae partial hybrid EC-1118 (Novo et al., 2010) contains a homologous FSY1 gene that is different from the S. eubayanus allele; it was also negative in tests.
Results with single-genome species and strains are unambiguous (Muir et al., 2011). With hybrids and strains with introgressions (Galeote et al., 2010; Naumova et al., 2011), results may be variable. For example, with S. pastorianus, which is a S. cerevisiae/S. eubayanus hybrid of recent origin (Dunn & Sherlock, 2008; Libkind et al., 2011), a positive result can be obtained for both the Seub and Scer primers (see Muir et al., 2011 for comparable results with Scer primers). However, results with isolates subject to intense selection in fermentations should be interpreted with caution. For example, loss of one homologous region by recombination or gene conversion will produce the pattern of only one parent, and this caveat will also be true of identification by single gene sequencing of hybrids. This was seen in one of the very earliest papers on rDNA sequencing (Peterson & Kurtzman, 1991) where the type strain of the hybrid S. pastorianus had sequences similar only to the type strain of S. bayanus (or as we now know it to be, S. eubayanus). We have seen negative results with Seub primers against S. pastorianus NCYC 453 (where the catalogue states ‘Sequence analysis gives 100% match to S. cerevisiae type strain’) and NCYC 679.1
Saccharomyces species-specific primers for the seven single-genome species are now available, and all seven pairs of primers can be used in multiplex format to identify their separate targets successfully. This works well when the number of primer pairs being used is limited. However, the number of different combinations rises rapidly with each extra primer pair – with seven pairs it is 105 – so the potential for false-positive reactions with out-groups becomes significant (although usually detectable; Harrison et al., 2011, figure 7).
We thank Chris Todd Hittinger for advice on sequence data, Gianni Liti for S. arboricola sequences, Steve James for advice on strains, Anna Mellors for technical support and the University of Bath for funding.