Microarray karyotyping of commercial wine yeast strains reveals shared, as well as unique, genomic signatures - PubMed (original) (raw)

Microarray karyotyping of commercial wine yeast strains reveals shared, as well as unique, genomic signatures

Barbara Dunn et al. BMC Genomics. 2005.

Abstract

Background: Genetic differences between yeast strains used in wine-making may account for some of the variation seen in their fermentation properties and may also produce differing sensory characteristics in the final wine product itself. To investigate this, we have determined genomic differences among several Saccharomyces cerevisiae wine strains by using a "microarray karyotyping" (also known as "array-CGH" or "aCGH") technique.

Results: We have studied four commonly used commercial wine yeast strains, assaying three independent isolates from each strain. All four wine strains showed common differences with respect to the laboratory S. cerevisiae strain S288C, some of which may be specific to commercial wine yeasts. We observed very little intra-strain variation; i.e., the genomic karyotypes of different commercial isolates of the same strain looked very similar, although an exception to this was seen among the Montrachet isolates. A moderate amount of inter-strain genomic variation between the four wine strains was observed, mostly in the form of depletions or amplifications of single genes; these differences allowed unique identification of each strain. Many of the inter-strain differences appear to be in transporter genes, especially hexose transporters (HXT genes), metal ion sensors/transporters (CUP1, ZRT1, ENA genes), members of the major facilitator superfamily, and in genes involved in drug response (PDR3, SNQ1, QDR1, RDS1, AYT1, YAR068W). We therefore used halo assays to investigate the response of these strains to three different fungicidal drugs (cycloheximide, clotrimazole, sulfomethuron methyl). Strains with fewer copies of the CUP1 loci showed hypersensitivity to sulfomethuron methyl.

Conclusion: Microarray karyotyping is a useful tool for analyzing the genome structures of wine yeasts. Despite only small to moderate variations in gene copy numbers between different wine yeast strains and within different isolates of a given strain, there was enough variation to allow unique identification of strains; additionally, some of the variation correlated with drug sensitivity. The relatively small number of differences seen by microarray karyotyping between the strains suggests that the differences in fermentative and organoleptic properties ascribed to these different strains may arise from a small number of genetic changes, making it possible to test whether the observed differences do indeed confer different sensory properties in the finished wine.

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Figures

Figure 1

Figure 1

Karyoscope views of S288C-S288C and _S. bayanus_-S288C microarray hybridizations. Microarray hybridizations were performed as described in the text. In both panels the S. cerevisiae laboratory strain S288C was used as a reference sample, labeled with Cy3 dye (green). In panel A, the Cy5-labeled (red) DNA was also S288C, giving a self-self hybridization. In panel B, the Cy-5 labeled DNA was S. bayanus. The labeled DNAs were competitively hybridized to spotted microarrays bearing full-length ORFs from the S288C S. cerevisiae strain; the data thus obtained is displayed here in graphical form as a karyoscope. Red bars indicate red:green ratios above 1.0 and are graphed on a log scale; green bars indicate red:green ratios below 1.0, also graphed on a log scale. The chromosomes are shown in each panel in numerical order with chromosome 1 at the top and chromosome 16 at the bottom, and are aligned by their centromeres, with their left arms extending to the left. Both karyoscopes are drawn to the same scale, i.e., the bar heights in panels A and B proportionally represent the same amplitude of change.

Figure 2

Figure 2

Changes in Gene Copy Number Shared by all Wine Strains: the "Commercial Wine Yeast Signature". A consensus plot pooling the results of all the wine strains relative to the lab strain S288C was generated by the program CGH-Miner [68]. This plot is similar to a karyoscope in that it displays values along each chromosome, and the chromosomes are shown in numerical order from top to bottom. However, in this plot, a determination of which regions were statistically significantly altered in copy number for each wine strain (i.e., averaged array values for each of the three isolates within a strain) relative to the laboratory S288C strain were determined. For each of these regions the number of wine strains showing a significant change were then added together to give a metric of how well-shared among the wine strains that particular change was. This is shown as tall red bars for significantly amplified regions among all wine yeast strains relative to the laboratory strain, and as tall green bars for significantly depleted regions in all the wine strains relative to the laboratory strain. Each gray "shadow line" above or below the chromosome represents 20% of the number of wine strains showing a significant change in that region (see legend). The group of changes comprising the tallest (>75%) red and green bars represents the "commercial wine strain signature"; these are listed in Table 2.

Figure 3

Figure 3

Wine strain karyoscopes. Microarray ratios from each of the three isolates of a given wine strain were averaged to yield an "average" microarray karyotype for each wine strain. Chromosomes 9 through 16 for the 4 strains are shown in panels A – D as follows: A. "Mont" = Montrachet (average of strains GSY1-3). B. "PDM" = Prise de Mousse (average of strains GSY10-12). C. "Champ" = Pasteur Champagne (average of strains GSY7-9). D. "Red" = French Red (average of strains GSY4-6). Circled regions highlight areas that vary in characteristic ways between the different wine strains. All karyoscopes are drawn to the same scale.

Figure 4

Figure 4

Karyoscopes of Chromosome 7 from individual Montrachet isolates. The larger black bar on the left, placed under the left end of chromosome 7 from the Lalvin Montrachet isolate (GSY1), shows a 37-ORF region that has been depleted (or deleted) with respect to the S288C laboratory strain and with respect to the chromosome 7's of each of the other two Montrachet isolates. The smaller black bar on the right, under the UCD522 (GSY3) chromosome, indicates a region of 3 ORFs (including MAL11 and MAL13) that are present at "wild-type" (S288C) copy number in the UCD522 strain but are depleted or deleted in the other two Montrachet strains. Note that this same MAL region is also present at "wild-type" copy number in UCD725, but is depleted or deleted in all remaining wine yeast strains. Again, all karyoscopes are drawn to the same scale.

Figure 5

Figure 5

Unique combinations of genomic differences between different strains. Panel A: Selected portions of the averaged data shown in Karyoscope form in Figure 3 are shown here in color-block form. To generate this figure the averaged data for all four wine strains were sorted for the Montrachet strain from highest green values (greatest negative number) to highest red values (greatest postitive number) and visualized as a "clustergram" using Java TreeView (note that the data were sorted, but however, were not clustered). The topmost (most green) 25 genes are shown in the top panel; the lower two panels show the sorted data surrounding two of the genes (AYT1 in the middle panel, YPL257W in the lowest panel) whose copy number varies greatly on a strain-to-strain basis. Red asterisks mark the genes whose copy numbers vary in unique combinations between the different strains. Panel B shows a matrix of gene copy number changes for each of the 12 wine strain isolates, along with the drug response profiles of each strain isolate at the bottom. Within the copy number data portion, a dark red cell with a "++" symbol indicates a red:green log ratio greater than 0.8, light red with "+" indicates a ratio between 0.2 and 0.8, and gray with "+/-" indicates a ratio between 0.2 and -0.2. Likewise dark green with "--" indicates a red:green log ratio less than -0.8, light green with "-" indicates a ratio between -0.2 and -0.8.

Figure 6

Figure 6

Drug resistance phenotypes of wine strains. Halo assays were performed as described in the text to test the response of the wine strains to various drugs. Panels A and B show photographs of the halos; on the far left of each panel are shown the halo assays for the WT-diploid (FY1679, a S288C-based diploid, see Methods), against which all other strains were compared. CYH = 500 ng cycloheximide; CTZ = 5 μg clotrimazole; SMM = 20 μg sulfomethuron methyl. Panel A: strains are as described in Methods and Table 1; "WT-Dip" refers to FY1679. Panel B: Strains are as described in Methods; "WT-Dip" refers to FY1679; "YPL257 Δ" to GSY28; "ayt1 Δ" to ScAYT1Δ, "pdr5-" to JG436; and "WT-Hap" to S288C. Panel C: Graphical results of halo assays. Annular area (i.e., area of total halo minus area of disc) was calculated for each halo, then expressed as log2 of the ratio of the annular area to that of the WT-Dip (FY1679) annular area.

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