Characterization of Malus Genotypes Within the Usda-Pgru Malus Germplasm Collection for Their Potential Use Within the Hard Cider Industry
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CHARACTERIZATION OF MALUS GENOTYPES WITHIN THE USDA-PGRU MALUS GERMPLASM COLLECTION FOR THEIR POTENTIAL USE WITHIN THE HARD CIDER INDUSTRY A Thesis Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Master of Science by Nathan Carey Wojtyna August 2018 © 2018 Nathan Carey Wojtyna ABSTRACT In the United States, hard cider producers lack access to apple genotypes (Malus ×domestica Borkh. and other Malus species) that possess higher concentrations of tannins (polyphenols that taste bitter and/or astringent) and acidity (described as having a sharp taste) than what is typically found in culinary apples. Utilizing the USDA-PGRU Malus germplasm collection, two projects were conducted to address these concerns. The first project characterized fruit quality and juice chemistry for a target population of 308 accessions with the goal of identifying accessions with desirable characteristics for hard cider production. The second project used the same sample population to explore the use of the Ma1 and Q8 genes as potential markers to predict the concentration of titratable acidity of cider apples. An initial target population of 308 accessions were identified and 158 accessions were assessed in 2017 for external and internal fruit characteristics along with juice chemistry. As per the Long Ashton Research Station (LARS) cider apple classification system where apples with tannin concentration (measured with the Löwenthal Permanganate Titration method) greater than 2.0 g×L-1 are classified as bitter, and those with a malic acid concentration greater than 4.5 g×L-1 are classified as sharp, 29% of the 158 accessions would be classified as bittersweet, 13% bittersharp, 28% sweet (neither bitter nor sharp), and 30% sharp. In addition, the polyphenol composition of 14 genotypes was measured using ultra high-performance liquid chromatography. and indicated that the variation in polyphenol levels measured by the Folin-Ciocalteu assay was largely due to procyanidins and phloretin compounds. In Chapter 3, the accessions phenotyped in the 2017 harvest season were also genotyped for the Ma1 and Q8 genes and fit to a linear model where measured titratable acidity was the response variable and the Ma, Q8 and combined Ma1 and Q8 genotypes were used as predictor variables in two separate models. An ANOVA indicates that the Ma1 gene was found to be a reliable predictor of fruit titratable acidity (P = < 0.0001), but the Q8 gene was not (P = 0.48). Combining both genes in the analysis was not a better predictor than using the Ma1 gene alone (P = 0.27). From there an estimated marginal means model, was able to demonstrate a series of non-overlapping 99% confidence intervals for the means titratable acidity concentrations of each Ma1 allele. Additionally, an estimated marginal means test of the Ma1 alleles was able to accurately predict the acidity classification as per the 4.5 g×L-1 threshold for the LARS cider apple classification system. Thus, a gene-based classification system of cider apples is proposed, with the titratable acidity levels defined by the Ma1 allele thresholds of 5.26 and 7.82 g×L-1. The advantage of a genetically based classification system is that it could be used for marker assisted breeding and it would not be subject to horticultural, seasonal, or geographic variability. Together, these two projects defined procedures for rapid phenotyping and genotyping of germplasm collections for addressing the needs of the emerging hard cider industry. Additionally, the characterization of cider apples for juice quality identified 49 bittersweet and 21 bittersharp genotypes that should be further evaluated for their suitability to perform in commercial orchards. II BIOGRAPHICAL SKETCH Nathan Wojtyna was born to Stephen Wojtyna and Lisa Carey-Michaud January 1994 and was also raised by his step-mother Denise Wojtyna and step-father Robert Michaud. As a child, Nathan Wojtyna was enamored with reading, in particular with books about the nature that came to life exploring the 300 acres of woods surrounding his home in Willington, Connecticut. After graduating from Edwin O. Smith High School in 2012, he attended the University of Connecticut where he majored in Horticulture and became passionate about applied industry research and added a double major in Resource Economics and minors in both Biotechnology and Environmental Economic Policy, graduating in 2016. Once he finished his studies at the University of Connecticut he moved to Ithaca, NY to conduct his master’s research at Cornell University. III To teachers who inspire their students and sow seeds of curiosity that last a lifetime. Also, to my friends and family, and especially Rachel Emily Knipple. You all supported me in countless ways in the pursuit of this degree. IV ACKNOWLEDGMENTS Scientific achievements and advancements are seldom the work of one individual and the work presented within this thesis is no exception. Many people contributed to this thesis and I hope I can give everyone the due recognition they deserve. First, to my principal advisor Dr. Gregory Peck for providing me with the financial support and moral coaching to always strive to produce the best possible research and for forging me into the young professional I have become today. Thank you, Greg, for your unwavering support and encouragement as I embark upon this journey to pursue my passions in life. This work would certainly not be possible without the work of Michael G. Brown, David Zakalik, Laura Dougherty, and Dr. Kenong Xu who have all gone above and beyond the call of duty to make this thesis possible. A special thank you also goes out to the past and present members of the Peck Lab, to Ian Merwin and to Gayle Volk, for all the feedback and collaboration in the developmental stages of this project. Also, a special thank you to the USDA- PGRU staff who work tirelessly to ensure the Malus germplasm collection continues to be an invaluable resource for research. Thank you to my graduate committee – Drs. Gavin Sacks and Thomas Chao for offering your insight and expertise throughout this research. Funding for this thesis work was provided by: Cornell University School of Integrated Plant Sciences – Horticulture Section, USDA Hatch Act funds, and the United States Association of Cider Makers (USACM). V TABLE OF CONTENTS Biographical Sketch .......................................................................................................................III Acknowledgments .......................................................................................................................... V Table of Contents .......................................................................................................................... VI Chapter One. Introduction ................................................................................................................. 1 References .......................................................................................................................... 10 Chapter Two. Characteristics of 158 Hard Cider Apple Genotypes in the USDA-PGRU Malus Germplasm Collection ....................................................................................................................... 12 Abstract ................................................................................................................................ 12 1.0. Introduction .......................................................................................................................... 13 2.0. Materials and Methods ......................................................................................................... 15 2.1. Targeted Accession Selection.................................................................................................. 15 2.2. Study Location and Sampling Design...................................................................................... 16 2.3. External Fruit Characteristics ................................................................................................. 18 2.4. Internal Fruit Characteristics ................................................................................................... 18 2.5. Juice Extraction ...................................................................................................................... 19 2.6. Juice Chemistry ...................................................................................................................... 19 2.7. Polyphenol Composition ......................................................................................................... 22 2.8. Statistical Analysis ................................................................................................................. 24 3.0. Results ................................................................................................................................... 24 3.1. Targeted Accession Selection ................................................................................................. 24 3.2. External Fruit Characteristics ................................................................................................. 25 3.3. Internal Fruit Characteristics ................................................................................................... 29 3.4. Juice Chemistry ...................................................................................................................... 31 3.5. Polyphenol