Canopy Position Influences on Gene Expression and Coffee Quality Bing Cheng M.Sc., B.Sc. A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Queensland Alliance for Agriculture and Food Innovation Abstract Coffee is one of the most valuable commodities exported around the world. Only two out of 125 species are highly valued in the genus Coffea, Arabica and Robusta coffee, accounting for 62% and 35% of the world coffee production. This project aimed to improve the understanding of the molecular and genetic basis of coffee quality through the detection of the upper and lower canopy position influences on coffee bean quality and gene expression. Selection from different parts of the plant might help to meet the increased threat from climate change and the demands of highly discerning consumers. Coffee quality is a very complicated trait depending on different perspectives. Phenotypic analysis was used to systematically access coffee quality through measurement of physical traits (including bean weight and size (length, width and thickness)), chemical components analysis (including determination of sucrose, trigonelline and caffeine content) and sensory evaluation to assess the aroma. To understand the influence of canopy position on coffee quality and gene expression, transcriptome analysis was conducted in this study. A reference transcriptome was generated for Arabica coffee using long read sequencing. To understand the dynamic of canopy position influences, three different ripening stages were selected. Upper canopy samples were used to understand the processes associated with coffee bean ripening and how the key components were accumulated in coffee beans through ripening. Thereafter, comparative transcriptome analysis was conducted to characterise the influence of canopy position. Higher sucrose, trigonelline and caffeine contents and greater aroma intensity were found in coffee beans from the lower canopy. According to transcriptome analysis, this increase resulted from increased expression of genes likely to be associated with these bean components and slower but longer maturation in coffee beans from the lower canopy. The slower and longer maturation was likely to be related with differential expression of genes associated with hormone inhibition and slower loss of photosynthesis. This study greatly improved understanding of molecular and genetic basis of coffee quality and canopy influences. Candidate genes from this study are potential for future breeding for high quality coffee. II Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. Publications included in this thesis 1. Cheng, B., Furtado, A., Smyth, H.E. and Henry, R.J., 2016. Influence of genotype and environment on coffee quality. Trends in Food Science & Technology, 57, pp.20-30. (This publication is extracted from chapter 2) Contributor Statement of contribution III Author Bing Cheng (Candidate) Conception and design (70%) Analysis and interpretation (80%) Drafting and production (80%) Author Robert J. Henry Conception and design (20%) Analysis and interpretation (20%) Drafting and production (10%) Author Agnelo Furtado Drafting and production (5%) Author Heather E. Smyth Drafting and production (5%) 2. Cheng, B., Furtado, A. and Henry, R.J., 2017. Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts. GigaScience, 6 (11), pp1-13. (This publication is incorporated in chapter 4) Contributor Statement of contribution Author Bing Cheng (Candidate) Conception and design (50%) Analysis and interpretation (90%) Drafting and production (80%) Author Robert J. Henry Conception and design (40%) Analysis and interpretation (10%) Drafting and production (15%) Author Agnelo Furtado Conception and design (10%) Drafting and production (5%) Cheng, B., Furtado, A. and Henry, R.J., 2018. The coffee bean transcriptome explains the accumulation of the major bean components through ripening. Scientific Reports, 8 (1) (This manuscript is incorporated in chapter 5) Contributor Statement of contribution Author Bing Cheng (Candidate) Conception and design (50%) Analysis and interpretation (80%) Drafting and production (80%) Author Robert J. Henry Conception and design (40%) Analysis and interpretation (10%) Drafting and production (10%) Author Agnelo Furtado Conception and design (10%) IV Analysis and interpretation (10%) Drafting and production (10%) Submitted manuscripts included in this thesis Cheng, B., Furtado, A., Smyth, H.E. and Henry, R.J. Analysis of Bean Phenotype and the Transcriptome from Different Canopy Positions Reveals Candidate Genes Influencing Coffee Quality. Submitted. (This manuscript is incorporated in chapter 3 and 6). Contributor Statement of contribution Author Bing Cheng (Candidate) Conception and design (50%) Analysis and interpretation (70%) Drafting and production (70%) Author Robert J. Henry Conception and design (30%) Analysis and interpretation (10%) Drafting and production (20%) Author Agnelo Furtado Conception and design (10%) Analysis and interpretation (10%) Drafting and production (5%) Author Heather E. Smyth Conception and design (10%) Analysis and interpretation (10%) Drafting and production (5%) Other publications during candidature No other publications. Contributions by others to the thesis No contributions by others. Statement of parts of the thesis submitted to qualify for the award of another degree No works submitted towards another degree have been included in this thesis. Research Involving Human or Animal Subjects No animal or human subjects were involved in this research. V Acknowledgements At this time I am about to finish, I really want to express my thanks to people who helped and made my PhD journey easier and enjoyable. My great thanks first goes to my supervisor, Professor Robert J. Henry, who was open- minded and accepted my PhD application when I was a beginner in bioinformatics, biotechnology and plant biology. Robert’s focus on the main question always keeps me on track. Without his patience and encouragement, I could not have finished this project so smoothly. The next thanks is to Dr. Agnelo Furtado, my co-supervisor. Agnelo taught me a lot in experiment design, record and data analysis. This makes my work more efficient and reproductive, and this training also benefits me in the non-academic field. I also appreciated my other co-supervisor, Dr Heather E. Smyth’s. She was very helpful in the phenotypic analysis, contributed critical ideas to the experiment, results and manuscripts. In addition, I am grateful to the useful feedbacks from my thesis panel, Dr. Tim O’ Hare, A/Prof. Bruce Topp and Dr. Glen Fox, as well as my thesis examiners. These feedbacks provide different perspectives of the results and are very useful to improve my thesis chapters. I thank Green Cauldron Coffee, Australia for providing the coffee fruit materials, pulping, husking and drying facilities; Poss Reading, Marta Brozynska, Adam Healey, Tiparat Tikapunya, Zhi Xian Lim, Ravi Nirmal, Nam Hoang and Hayba Badro for assistance with sample harvest and Poss Reading and Erli Wang for coffee sample processing. I thank the Wenny Bekti Sunarharum, Hue Tran and Tom Kukhang, who shared their knowledge in coffee. I thank the good feedbacks for Chapter six from Tom Kukhang, Douglas Soltis and Pam Soltis. I thank the critical feedbacks to my manuscripts from the anonymous peer- reviewers, Sandeep Chakraborty and Stephanie Bocs. Instrumental thanks go to Health Food Science Precinct (HFSP) of Department of Agriculture and Fisheries and the Research Computing Centre (RCC) of The University of Queensland for the phenotypic analysis. Special acknowledgement goes to Kent Fanning (Department of Agriculture and Fisheries, Queensland) and Igor Makunin (RCC), Kevin Smyth (RCC) and Erli Wang (School of Mathematics and Physics) from the University of Queensland.