Location: Plant, Soil and Nutrition Research
2023 Annual Report
Objectives
Objective 1. Develop new knowledge of the genes and regulatory interactions influencing the ripening and nutritional pathways of fruits and vegetables for consumers.
Sub-objective 1A: Identify novel genes regulating fruit ripening, nutrition, and shelf-life traits using tissue-focused characterization.
Sub-objective 1A.1: Phenotyping, transcriptional profiling, and sequencing of a tomato diversity core collection as a resource for exploring questions of fruit biology.
Sub-objective 1A.2: Identify gene structural variations associated with gene expression.
Sub-objective 1A.3: Functionally validate putative regulatory SVs via gene editing.
Sub-objective 1B: Define the role of transcriptional regulators and their homologs in the control of ripening, nutrition, shelf-life, and quality traits.
Sub-objective 1B.1: Genetic analysis of transcription factor homolog function.
Sub-objective 1B.2: Development of a tomato predicted protein structure database and analysis of protein interaction potential.
Sub-objective 1C: Isolation of the genes that control carotenoid accumulation in fruit and vegetables
Sub-objective 1D: Regulatory mechanisms underlying carotenoid and other nutrient accumulation
Objective 2. Optimize techniques for the extraction and quantitation of nutritional metabolites from micro-scale crop tissue samples.
Objective 3. Characterize at the whole genome level off-targeting of genome editing and methylome editing technologies.
Sub-objective 3.1: Assessment of off-targeting in gene-edited tomato lines.
Sub-objective 3.2: Development of targeted methylation edited tomato lines.
Sub-objective 3.3: Assessment of proteome changes in gene-edited tomato lines.
Approach
Molecular, genetic, and genomics strategies will be deployed to advance understanding of the molecular basis of fruit and vegetable quality, nutrition, and storability. Resulting genes, genetic diversity and metabolic insights related to these traits will be applied toward crop improvement. We will take advantage of existing germplasm in the form of mutant/variant lines, diversity panels, recombinant inbred and wild species introgression lines to identify genes underlying fruit and vegetable quality, storability and nutritional content. Candidate genes will be isolated, sequenced, and characterized for gene expression attributes in addition to allelic variation that will be correlated with trait and/or metabolic outputs. Functional analyses of genes will be undertaken for candidate quality, storage and nutrition impacting genes through gene editing and via correlation with trait variation resulting from natural genetic diversity. We will explore potential for translation of insights from model crop systems to additional crop species. Better understanding of processes underlying fruit and vegetable quality will facilitate design of targeted molecular strategies to improve crop quality attributes in both primary experimental crop systems and targets of translational biology. In developing gene edited and transgenic lines we will also create a resource to explore at the genome, epigenome and metabolome levels biotechnology risk, with an emphasis on gene-editing approaches that are of wide experimental, commercial and consumer interest.
Progress Report
Due to the start of the project four months prior to this report, none of the objective have been met but progress is being made on all. Our tomato diversity panel is currently growing in a replicated field trial in Freeville, New York, with tissue harvest and RNA extraction to begin soon. AlphaFold 2 has been used by others to create predicted structures for all 35,000 tomato genes with this data in the public domain. We are using this information to predict interactions with folate and among transcriptional regulators involved in ripening. B gene isolation is in progress and candidate transcription factors for Or regulation are in hand for functional analysis. Improved extraction protocols are being developed with significant progress in reliable and quantitative analysis of folates achieved to date. Plant materials for biotech risk analyses are being grown and constructs developed for addressing epigenome modifications. All milestones are on track to be substantially or fully met by the end of the first year since project initiation.
Accomplishments
