Fruit Crispness Retention of ‘Honeycrisp’ Apple and its Progeny A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF THE UNIVERSITY OF MINNESOTA BY Hsueh-Yuan Chang IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Cindy B. S. Tong July 2020 © Hsueh-Yuan Chang 2020 Acknowledgements Let me first express my gratitude to Dr. Cindy Tong. Without her, I probably would not have had the opportunity to pursue a Ph.D. degree. Cindy always believed in my ability and gave me the freedom to explore and develop my research passion. It has been a wonderful journey to work with Cindy on different aspects of postharvest research. I am grateful to my committee members: Dr. Zata Vickers, Dr. Jim Luby, Dr. Jim Bradeen and Dr. Matt Clark. They have been very generous with their time and advice. Many thanks for the guidance on this project and the comments on the publications. I have learned so much about being a scientist. There are many professors and researchers who provided various support to this project. I thank Dr. Joshua Baller, Dr. Thomas Kono, and Dr. Nelson Garcia for bioinformatic advice and help with developing the pipeline for RNA-Seq data analyses, I also like to thank Dr. Nicholas Howard and Dr. Soon Li Teh for sharing their knowledge and experience in QTL analyses. Furthermore, I thank Dr. Camila Alves for help in conducting qRT-PCR experiment, Jenna Brady for managing the sensory evaluation of apple fruit, Dr. Meng-Hsuan Wu for statistical support, and Dr. Theodore Labuza for use of texture analyzer and his lab. Lastly, I want to thank my colleagues in the office 358, the postharvest lab, and the fruit breeding lab in the Department of Horticultural Science for their valuable discussion and selfless support over the years. i Abstract Crispness retention is a unique postharvest trait that ‘Honeycrisp’ apple possesses. With adequate temperature and moisture conditions, ‘Honeycrisp’ fruit retain their highly crisp texture during long-term storage. This research project aims to extend understanding of the molecular mechanisms underlying crispness retention through studying a breeding population derived from ‘Honeycrisp’ x MN1764. In Chapter1, current knowledge regarding postharvest textural changes in apple fruit was reviewed. In Chapter 2, sensory evaluation and instrumental methods were applied to quantify fruit crispness of the breeding population. In Chapter 3, transcriptomes of the selected progeny individuals differed in their ability to retain postharvest crispness were compared to identify genes associated with crispness retention. By combining a genetically-related apple population, an improved phenotyping method for measuring fruit crispness, and transcriptomic analyses (RNA-Seq and nCounter®), we were able to identify novel candidate genes for crispness retention of ‘Honeycrisp’ fruit. ii Table of Contents Acknowledgements .............................................................................................................. i Abstract ............................................................................................................................... ii List of Tables ...................................................................................................................... v List of Figures ................................................................................................................... vii Chapter 1: Literature Review .............................................................................................. 1 1.1 Introduction .......................................................................................................... 1 1.2 Crispness and firmness ......................................................................................... 1 1.3 Fruit cell walls ...................................................................................................... 2 1.4 Cell wall-modifying enzymes .............................................................................. 3 1.5 Chromosome regions............................................................................................ 7 1.6 Transcriptomic approaches .................................................................................. 8 1.7 Honeycrisp ......................................................................................................... 11 1.8 Conclusion .......................................................................................................... 13 Chapter 2: Correlations Between Sensory and Instrumental Crispness of a ‘Honeycrisp’ Apple Breeding Population ............................................................................................... 16 2.1 Introduction ........................................................................................................ 16 2.2 Materials and Methods ....................................................................................... 20 2.2.1 Plant Materials ............................................................................................ 20 2.2.2 Sensory Evaluation ..................................................................................... 20 2.2.3 Instrumental Tests ....................................................................................... 22 2.2.4 Data Analyses ............................................................................................. 23 2.3 Results ................................................................................................................ 26 2.3.1 Sensory Crispness ....................................................................................... 26 2.3.2 Instrumental Measurements ........................................................................ 27 2.3.3 Correlations between Sensory Crispness and Instrumental Measures ........ 28 2.3.4 Application of Principal Component Analysis to Instrumental Measures.. 29 2.3.5 Multiple Linear Regression Models............................................................ 30 2.4 Discussion .......................................................................................................... 30 2.4.1 Comparison of the Instrumental Methods................................................... 30 iii 2.4.2 Identification of Individuals with Fruit that Retain or Lose Crispness ....... 32 2.4.3 Instrumental Prediction of Crispness and Change in Crispness ................. 33 2.5 Conclusions ........................................................................................................ 34 Chapter 3: Transcriptome analyses identifying genes associated with crispness retention of ‘Honeycrisp’ fruit and its progeny................................................................................ 46 3.1 Introduction ........................................................................................................ 47 3.2 Materials and Methods ....................................................................................... 47 3.2.1 Plant materials ............................................................................................. 49 3.2.2 RNA sample preparation and RNA sequencing ......................................... 50 3.2.3 Differential expression analysis .................................................................. 51 3.2.4 Gene validation using NanoString nCounter® and qRT-PCR .................... 52 3.3 Results ................................................................................................................ 53 3.3.1 Phenotype and transcriptome variations among the individuals................. 54 3.3.2 Functional analyses of differentially-expressed genes ............................... 54 3.3.3 The expression patterns of auxin- and ethylene-related genes ................... 56 3.3.4 The expression patterns of cell wall-related genes ..................................... 59 3.3.5 RNA-Seq results validation using nCounter® technology .......................... 59 3.4 Discussion .......................................................................................................... 61 3.4.1 Fruit ripening and crispness retention ......................................................... 61 3.4.2 Cell wall-related genes and crispness retention .......................................... 61 3.5 Conclusion .......................................................................................................... 66 Bibliography ..................................................................................................................... 93 Appendices ...................................................................................................................... 106 iv List of Tables Table 2-1 Sensory texture attributes and their definitions. .............................................. 35 Table 2-2 Instrumental measures generated by puncture, mechanical-acoustic, and snapping tests. ................................................................................................................... 36 Table 2-3 Sensory crispness, puncture force (PF), force linear distance (FLD), maximum force (F2), and maximum acoustic pressure (AUX1) of the 20 individuals and the two parents from the ‘Honeycrisp’ x MN1764 family at harvest and after 8-week storage. Statistical significance of differences (diff.) between fresh and stored fruit of each individual were determined by ANOVA. ......................................................................... 37 Table 2-4 Correlation coefficients (r) between