Dominance of indigenous Saccharomyces uvarum in spontaneous wine fermentations conducted in the Okanagan Valley by Garrett McCarthy B.Sc., The University of British Columbia, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2019 © Garrett McCarthy, 2019 i The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Dominance of Saccharomyces uvarum in spontaneous wine fermentations conducted in the Okanagan Valley submitted by Garrett McCarthy in partial fulfillment of the requirements of the degree of Master of Science Dr. Dan Durall, Irving K. Barber School of Arts and Sciences Supervisor Dr. Vivien Measday, Wine Research Centre, Faculty of Land and Food Systems Supervisor Dr. Louise Nelson, Irving K. Barber School of Arts and Sciences Supervisory Committee Member Dr. Sepideh Pakpour, School of Engineering University Examiner ii Abstract The majority of wines are produced by inoculated alcoholic fermentations using commercialized strains of Saccharomyces cerevisiae, yet there is a growing trend in winemaking to perform spontaneous fermentations, which rely on microbiota present on grape berries, and/or on winery surfaces and equipment. An advantage for spontaneous over inoculated fermentation is a more complex sensory profile due to a wider range of metabolites from differing yeast species, which may help to define the regional identity of the wine. Once the spontaneous fermentation progresses into the mid and late stages of fermentation, ethanol-tolerant S. cerevisiae strains usually dominate. Contrarily, previous studies in our lab showed that an indigenous yeast, Saccharomyces uvarum, can dominate over S. cerevisiae during spontaneous Okanagan Chardonnay fermentations. My objectives were to: 1) determine if S. uvarum was again part of the fungal community in spontaneous Chardonnay fermentations; and if so; 2) develop an improved classification method for S. uvarum strains; 3) determine the degree of genetic diversity of the S. uvarum populations in spontaneous Chardonnay fermentations, which differ in their grape origin; and 4) determine whether S. uvarum was part of the fungal community on grapes from different vineyards of the Okanagan Valley wine region. Using Illumina high-throughput sequencing, I found S. uvarum was again dominant over S. cerevisiae in all winery fermentations, similar to findings in 2015 from spontaneous fermentations in the same winery. Using culture-dependent methods, I performed S. uvarum strain analysis with a novel, 11-loci microsatellite multiplex screen, and used Bruvo genetic distance to classify multi- locus genotypes into strains. I identified over 200 multi-locus genotypes and classified them into over 50 unique strains from winery fermentations that differed significantly in composition between treatments comprising grapes from two Chardonnay vineyards. As well, high- throughput amplicon sequencing revealed the presence of S. uvarum throughout all four vineyard iii locations spread across the Okanagan Valley wine region. My results indicate there may be commercial interest in using S. uvarum as a potential alternative to S. cerevisiae, which is typically the species used in winery fermentations. iv Lay Summary This thesis provides evidence that a yeast called Saccharomyces uvarum found from the winery environment can dominate alcoholic fermentations in the Okanagan Valley, even outcompeting the typical yeast used called Saccharomyces cerevisiae. The S. uvarum found in the Okanagan was also unexpectedly genetically diverse as compared with what has been seen before in the literature. Grapes were sourced from two different vineyards and followed into the same winery as the two treatments, where the fermentations were taken over mainly by the resident S. uvarum yeast at the winery. There were differences in S. uvarum strain composition between the two treatments. I also found that S. uvarum was present on grapes in four vineyards (two were the vineyards followed into the winery), which were spread across the Okanagan Valley wine region. Seeing how well it can complete the fermentations, certain strains of S. uvarum may be an alternative choice to S. cerevisiae strains when winemakers select yeast starters for Chardonnay fermentations. v Preface I was responsible for implementing wine fermentations according to the experimental design as well as collecting all the samples from Mission Hill Family Estate Winery and from four vineyards during the 2017 vintage for fungal community analysis. The fungal identification through culture-dependent and culture-independent methods were performed in the molecular lab at the University of British Columbia’s Okanagan campus. I was responsible for the majority of data collection from the winery and vineyard experimental designs, statistical analysis interpretation and writing the thesis, under the supervision of my co-supervisors Dr. Daniel Durall and Dr. Vivien Measday. My supervisory committee member Dr. Louise Nelson also reviewed this thesis. Brianne Newman assisted with culture-dependent methods and collection of vineyard samples. Sydney Morgan assisted with culture-independent, high-throughput sequencing preparation, and analysis of the sequenced reads. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ..................................................................................................................................x List of Figures .............................................................................................................................. xii Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv 1 Introduction .................................................................................................................................1 1.1 Alcoholic fermentation of grape juice ................................................................................... 2 1.1.1 Inoculated fermentation ................................................................................................. 2 1.1.2 Spontaneous fermentation .............................................................................................. 3 1.2 Saccharomyces uvarum ......................................................................................................... 5 1.2.1 Saccharomyces uvarum and the Saccharomyces sensu stricto ...................................... 5 1.2.2 Introgressions into S. uvarum strains ............................................................................. 6 1.2.3 Holoarctic Saccharomyces uvarum history and geography ........................................... 7 1.3 Physiological differences between S. uvarum and S. cerevisiae ........................................... 8 1.3.1 Psychrotrophic nature of S. uvarum ............................................................................... 8 1.3.2 S. uvarum and S. cerevisiae influence on wine body sensory characters ...................... 9 1.3.3 S. uvarum and S. cerevisiae influence on wine aroma sensory characters .................. 11 1.4 Identifying and characterizing S. uvarum through both species and strain typing analysis 12 1.4.1 Genotyping of S. uvarum strains .................................................................................. 12 vii 1.4.2 High-throughput amplicon sequencing of yeast species in grape and spontaneous wine fermentations......................................................................................................................... 14 1.5 Thesis objectives and hypotheses ........................................................................................ 16 1.5.1 Background to objectives and hypotheses ................................................................... 16 1.5.2 Objectives and hypotheses ........................................................................................... 16 2 High genotypic diversity and dominance of indigenous Saccharomyces uvarum in spontaneous Chardonnay fermentations conducted at an Okanagan Valley winery ...........20 2.1 Background ......................................................................................................................... 20 2.2 Materials and Methods ........................................................................................................ 21 2.2.1 Spontaneous fermentation experimental design in the MHFE winery ........................ 21 2.2.2 High throughput amplicon sequencing (HTAS) for analysis of species community composition ..........................................................................................................................