Whole genome sequencing of Canadian Saccharomyces cerevisiae strains isolated from spontaneous wine fermentations reveals a new Pacific West Coast Wine clade.

Vineyards in wine regions around the world are reservoirs of yeast with oenological potential. Saccharomyces cerevisiae ferments grape sugars to ethanol and generates flavor and aroma compounds in wine. Wineries place a high-value on identifying yeast native to their region to develop a region-specific wine program. Commercial wine strains are genetically very similar due to a population bottleneck and in-breeding compared to the diversity of S. cerevisiae from the wild and other industrial processes. We have isolated and microsatellite-typed hundreds of S. cerevisiae strains from spontaneous fermentations of grapes from the Okanagan Valley wine region in British Columbia, Canada. We chose 75 S. cerevisiae strains, based on our microsatellite clustering data, for whole genome sequencing using Illumina paired-end reads. Phylogenetic analysis shows that British Columbian S. cerevisiae strains cluster into 4 clades: Wine/European, Transpacific Oak, Beer 1/Mixed Origin, and a new clade that we have designated as Pacific West Coast Wine. The Pacific West Coast Wine clade has high nucleotide diversity and shares genomic characteristics with wild North American oak strains but also has gene flow from Wine/European and Ecuadorian clades. We analyzed gene copy number variations to find evidence of domestication and found that strains in the Wine/European and Pacific West Coast Wine clades have gene copy number variation reflective of adaptations to the wine-making environment. The "wine circle/Region B", a cluster of 5 genes acquired by horizontal gene transfer into the genome of commercial wine strains is also present in the majority of the British Columbian strains in the Wine/European clade but in a minority of the Pacific West Coast Wine clade strains. Previous studies have shown that S. cerevisiae strains isolated from Mediterranean Oak trees may be the living ancestors of European wine yeast strains. This study is the first to isolate S. cerevisiae strains with genetic similarity to nonvineyard North American Oak strains from spontaneous wine fermentations.

Grape-associated fungal community patterns persist from berry to wine on a fine geographical scale.

Wine grape fungal community composition is influenced by abiotic factors including geography and vintage. Compositional differences may correlate with different wine metabolite composition and sensory profiles, suggesting a microbial role in the shaping of a wine's terroir, or regional character. While grape and wine-associated fungal community composition has been studied extensively at a regional and sub-regional scale, it has not been explored in detail on fine geographical scales over multiple harvests. Over two years, we examined the fungal communities on Vitis Vinifera cv. Pinot noir grape berry surfaces, in crushed grapes, and in lab spontaneous fermentations from three vineyards within a < 1 km radius in Canada's Okanagan Valley wine region. We also evaluated the effect of winery environment exposure on fungal community composition by sampling grapes crushed and fermented in the winery at commercial scale. Spatiotemporal community structure was evident among grape berry surface, crushed grape and fermentation samples, with each vineyard exhibiting a distinct fungal community signature. Crushed grape fungal populations were richer in fermentative yeast species compared to grape berry surface fungal populations. Our study suggests that, as on a regional level, fungal populations may contribute to fine-scale -terroir,' with significant implications for single-vineyard wines.

An indigenous Saccharomyces uvarum population with high genetic diversity dominates uninoculated Chardonnay fermentations at a Canadian winery.

Saccharomyces cerevisiae is the primary yeast species responsible for most fermentations in winemaking. However, other yeasts, including Saccharomyces uvarum, have occasionally been found conducting commercial fermentations around the world. S. uvarum is typically associated with white wine fermentations in cool-climate wine regions, and has been identified as the dominant yeast in fermentations from France, Hungary, northern Italy, and, recently, Canada. However, little is known about how the origin and genetic diversity of the Canadian S. uvarum population relates to strains from other parts of the world. In this study, a highly diverse S. uvarum population was found dominating uninoculated commercial fermentations of Chardonnay grapes sourced from two different vineyards. Most of the strains identified were found to be genetically distinct from S. uvarum strains isolated globally. Of the 106 strains of S. uvarum identified in this study, four played a dominant role in the fermentations, with some strains predominating in the fermentations from one vineyard over the other. Furthermore, two of these dominant strains were previously identified as dominant strains in uninoculated Chardonnay fermentations at the same winery two years earlier, suggesting the presence of a winery-resident population of indigenous S. uvarum. This research provides valuable insight into the diversity and persistence of non-commercial S. uvarum strains in North America, and a stepping stone for future work into the enological potential of an alternative Saccharomyces yeast species.

Characterization of Sub-Regional Variation in Saccharomyces Populations and Grape Phenolic Composition in Pinot Noir Vineyards of a Canadian Wine Region.

Wine is a product of grape juice fermentation by yeast. Terroir is a term that encompasses all environmental factors and interactions at a specific geographical site, resulting in the development of regional-specific microbial strains and grape metabolites. In this study we determine the distribution of vineyard-associated wine yeast strains and characterize the flavonoid profile of Pinot Noir grapes among 3 sub-regions in the Okanagan Valley (OV), a major wine region in British Columbia, Canada. Pinot Noir grape samples were collected from 13 vineyards among 3 sub-regions of the OV, namely Kelowna (KE), Naramata-Penticton (NP) and Oliver-Osoyoos (OO), within a week prior to the winery harvesting date in 2016 and 2017. A total of 156 spontaneous Pinot Noir fermentations were conducted and vineyard-associated Saccharomyces strains were isolated from fermentations that reached two-thirds sugar depletion. Using microsatellite genotyping, we identified 103 Saccharomyces cerevisiae strains and 9 Saccharomyces uvarum strains. We also identified Saccharomyces paradoxus in one vineyard using ITS sequencing. We developed a microsatellite database of 160 commercial S. cerevisiae strains to determine the identity of the isolated strains and we include the database herein. Commercial strains were widely distributed across the three sub-regions. Forty-two of our 103 S. cerevisiae strains were equivalent or highly similar to commercial strains whereas the remaining 61 were considered as 'unknown' strains. Two S. uvarum strains were previously isolated in other OV studies and none matched the S. uvarum commercial strain BMV58. S. cerevisiae population structure was driven by sub-region, although S. cerevisiae populations did not differ significantly across vintages. S. uvarum and S. paradoxus were only identified in the 2017 vintage, demonstrating dynamic wine yeast populations between vintages. We found that the flavonoid profile of Pinot Noir grapes from the same 13 vineyards was also affected by sub-regional terroir. The anthocyanin content was lower and the proportion of methoxylated anthocyanins and flavonols was higher in Pinot Noir grapes from OO, the warmer sub-region as compared to KE, the cooler sub-region. Our study demonstrates that both yeast populations and metabolites associated with the Pinot Noir variety have sub-regional variation within a viticultural area.

Impact of Commercial Strain Use on Saccharomyces cerevisiae Population Structure and Dynamics in Pinot Noir Vineyards and Spontaneous Fermentations of a Canadian Winery.

Wine is produced by one of two methods: inoculated fermentation, where a commercially-produced, single Saccharomyces cerevisiae (S. cerevisiae) yeast strain is used; or the traditional spontaneous fermentation, where yeast present on grape and winery surfaces carry out the fermentative process. Spontaneous fermentations are characterized by a diverse succession of yeast, ending with one or multiple strains of S. cerevisiae dominating the fermentation. In wineries using both fermentation methods, commercial strains may dominate spontaneous fermentations. We elucidate the impact of the winery environment and commercial strain use on S. cerevisiae population structure in spontaneous fermentations over two vintages by comparing S. cerevisiae populations in aseptically fermented grapes from a Canadian Pinot Noir vineyard to S. cerevisiae populations in winery-conducted fermentations of grapes from the same vineyard. We also characterize the vineyard-associated S. cerevisiae populations in two other geographically separate Pinot Noir vineyards farmed by the same winery. Winery fermentations were not dominated by commercial strains, but by a diverse number of strains with genotypes similar to commercial strains, suggesting that a population of S. cerevisiae derived from commercial strains is resident in the winery. Commercial and commercial-related yeast were also identified in the three vineyards examined, although at a lower frequency. There is low genetic differentiation and S. cerevisiae population structure between vineyards and between the vineyard and winery that persisted over both vintages, indicating commercial yeast are a driver of S. cerevisiae population structure. We also have evidence of distinct and persistent populations of winery and vineyard-associated S. cerevisiae populations unrelated to commercial strains. This study is the first to characterize S. cerevisiae populations in Canadian vineyards.