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Naturally Occurring Variation in the Promoter of the Chromoplast-Specific Cyc-B Gene in Tomato Can Be Used to Modulate Levels of Β-Carotene in Ripe Tomato Fruit

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Naturally Occurring Variation in the Promoter of the Chromoplast-Specific Cyc-B Gene in Tomato Can Be Used to Modulate Levels of Β-Carotene in Ripe Tomato Fruit Naturally occurring variation in the promoter of the chromoplast-specific Cyc-B gene in tomato can be used to modulate levels of β-carotene in ripe tomato fruit THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Caleb James Orchard Graduate Program in Horticulture and Crop Science The Ohio State University 2014 Thesis Committee: Dr. David Francis, “Advisor” Dr. Leah McHale Dr. Joseph Scheerens Dr. Steven Schwartz Copyright by Caleb James Orchard 2014 Abstract β-carotene in tomato is an important carotenoid for human health due to its pro- vitamin A activity. Although many carotenoids exhibit provitamin A activity and are available in food crops, Vitamin A deficiency remains the leading cause of preventable blindness in many developing countries. In tomato (S. lycopersicum, L.), the B gene (Cyc- B) encodes a chromoplast-specific lycopene-β-cyclase that converts trans-lycopene to β- carotene. Prior research suggests that DNA sequence variation in the promoter of B may be responsible for the high β-carotene Beta phenotype. We examined the carotenoid profiles of vintage and contemporary tomato varieties to identify sources of high β- carotene. Red tomatoes had a range from 0.2 – 0.97 mg/100 g fresh weight of β-carotene, while several orange-fruited varieties had 1.67 – 4.0 mg/100 g. The non-transcribed region 5′ to the B gene (promoter) contains significant nucleotide variation, with eleven unique haplotypes across 1850 bp of sequence. Sequence analysis suggested that the B promoter was derived from wild tomato species. Association mapping and non- parametric statistical approaches suggest two single nucleotide polymorphisms (SNPs) as the most likely cause(s) of high β-carotene, presumably through their influence on transcription of the gene. A marker-assisted backcross breeding scheme leveraging genome-wide SNPs was used to rapidly develop a series of genetic resources containing different alleles of the B promoter in a uniform genetic background. Replicated field trials demonstrated that distinct alleles can be used to modulate the levels of β-carotene in ii tomato. These genetic resources are available to develop β-carotene enriched food products or to study dietary adsorption and utilization of carotenoids in the food matrix. Furthermore, studying the basis of variation in carotenoid biosynthesis in general, and specifically β-carotene, provides a clearer understanding of biochemical regulation and phenotypic variation in plants. iii Acknowledgments Many people contributed to the formation of this thesis and the findings presented therein. I would like to thank the following people for their kind support and advice: my lab group including Deborah Liabeuf, Nancy Haurachi-Morejon, Nico Lara, Andrew Kruezman, Brayton Orchard, Gabriel Abud, Marcela Andrade, Eka Sari and Benard Eriku for their help in data generation, visualization and advice; Troy Aldrich and Jiheun Cho for taking care of my plants in the greenhouse and field; Kesia Harztler and Mike Devault for overseeing greenhouse operations; the field crews in Wooster and in Fremont led by Bruce Williams and Matt Hoeflich for maintaining my plants in the field; Jessica Cooperstone and the entire Schwartz Food Science lab for their hospitality and assistance in carotenoid profiling; members of the MCIC at OARDC for their assistance in sequencing and genotyping; my committee members Dr.’s Leah McHale, Steven Schwartz and Joseph Scheerens for their willingness to serve on my committee and for their time and effort in reviewing this thesis; Sung-Chur Sim for the excellent mentorship he provided early in my Master’s experience; and my family for their constant support and encouragement. Finally, I would like to thank my advisor, Dr. David Francis, for his continued support, mentorship and kindness throughout my time in graduate school. This thesis was only made possible by the kind support and advice of these people. iv Vita June, 2007 ......................................................Loudonville High School May 2011 ......................................................B.S. Biology, Political Science, Grove City College 2012 to present ..............................................Graduate Research Assistant, Department of Horticulture and Crop Science, The Ohio State University Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita .......................................................................................................................................v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1: Introduction ........................................................................................................1 Carotenoids ..............................................................................................................1 β-carotene .................................................................................................................2 Carotenoid biosynthesis in tomato ...........................................................................4 Classical characterization of the B locus .................................................................7 Molecular characterization of the B locus ...............................................................8 Exploring allelic variation through sequencing .......................................................8 Backcrossing ..........................................................................................................10 Marker-assisted backcrossing ................................................................................10 Rationale and Significance ....................................................................................14 Approach ................................................................................................................16 Project Aim ............................................................................................................20 Chapter 2: Sequencing the promoter of the chromoplast-specific Cyc-B gene in tomato reveals novel high β-carotene alleles and putative functional mutations ..........................21 Acknowledgments..................................................................................................21 Abstract ..................................................................................................................22 Introduction ............................................................................................................23 Materials and Methods ...........................................................................................25 vi Results ....................................................................................................................31 Discussion ..............................................................................................................35 Chapter 3: Naturally occurring variation in the promoter of the chromoplast-specific Cyc- B gene in tomato can be used to modulate levels of β-carotene in ripe tomato fruit .........46 Acknowledgments..................................................................................................46 Abstract ..................................................................................................................47 Introduction ............................................................................................................48 Materials and Methods ...........................................................................................52 Results ....................................................................................................................58 Discussion ..............................................................................................................61 Chapter 4: Conclusion........................................................................................................72 References ..........................................................................................................................76 Appendix A: SNPs used for background genome selection in BC1 ..................................85 Appendix B: SNPs used for background genome selection in BC2...................................88 vii List of Tables Table 2.1: Carotenoid profiling by HPLC of 29 orange and red-fruited tomato accessions. ..........................................................................................................................39 Table 3.1: Distribution of percent recurrent parent genome (OH8245) in sub-populations in BC1 and BC2 generations ...............................................................................................67 Table 3.2: Sources of variation in β-carotene content in ripe tomato fruit over two years ............................................................................................................................................68 viii List of Figures Figure 1.1: Simplified depiction
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