Project : USDA ARS

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Research Project: Genetics, Epigenetics, Genomics, and Biotechnology for Fruit and Vegetable Quality

Location: Plant, Soil and Nutrition Research

2020 Annual Report

Objectives
Objective 1: Identify loci and functionally characterize underlying genes that contribute to fruit and vegetable shelf-life, appearance, flavor, texture, and nutritional quality by characterizing cultivated and wild species diversity so as to develop a better understanding of corresponding trait biology and to develop new molecular tools for breeding. (See uploaded postplan for sub-objectives) Objective 2: Generate genome-scale DNA sequence data, gene expression profiles, proteomic and metabolite data of fruit and vegetable crops for facilitating trait discovery and trait improvement. (See uploaded postplan for sub-objectives) Objective 3: Develop and test models for the regulation of fruit and vegetable development and quality traits at the genome level that incorporate epigenome dynamics and epigenetic regulators. (See uploaded postplan for sub-objectives) Objective 4: Develop and utilize new advanced analytical approaches to characterize fruit and vegetable proteins and chemical metabolites, their modifications and interactions, via targeted and genome-scale methodologies. (See uploaded postplan for sub-objectives) Objective 5: Develop, test, and thoroughly analyze at the whole genome level gene editing technologies in tomato for use in enhancing nutrient levels and shelf-life, and in selected high value crops for use in breeding and research. (See uploaded postplan for sub-objectives)

Approach
The overall approach of this project will be use of molecular, genetic and genomics approaches to address our objectives centered on advancing our understanding of fruit and vegetable quality and deploying said knowledge toward crop improvement. We will take advantage of existing germplasm in the form of mutant/variant lines and segregating populations and/or wild species introgression lines to identify genes underlying fruit and vegetable quality 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 will be undertaking for candidate quality and nutrition impacting genes through identification and development, respectively, of chemical/natural or transgenic mutations. In some instances, we will test potential for translation of insights from model and crop systems studies to additional crop and stable crop species. Better understanding of processes underlying fruit and vegetable quality will facilitate design of molecular strategies to improve crop quality attributes in both primary experimental crop systems and targets of translational biology. Through these undertakings, we will develop transgenic and gene edited lines to address gene function. We will further utilize said lines and additional lines developed as controls to assess the nature and degree of genome changes resulting from transformation or gene editing and the extent of possible biotechnological risk, if any.

Progress Report
Exploring the potential of tomato wild species genetic diversity: The project involves assessment of tomato genetic diversity toward understanding fruit development, ripening and nutritional quality while simultaneously using resulting materials for biotechnology risk assessment with an emphasis on emerging gene editing approaches. Previously, researchers grew tomato populations resulting from crosses of cultivated and wild species. These populations are both recombinant inbred (RIL) and introgression line (IL) populations – a key feature being that they are inbred/true breeding, allowing low resolution mapping of observed traits and recovery of identical lines for future experiments. This year involved phenotypic analysis of resulting fruit, especially for nutritional chemicals including carotenoids and their derivatives, folic acid and initiation of efforts to explore flavonoids. Flavonoid chemistry is more complex so we are exploring ways of developing/optimizing protocols to more efficiently extract and analyze these compounds. We continue to focus on a population derived from a cross between tomato and its most diverged sexually compatible relative, Solanum lycopersicoides, which is adapted to extreme cold and drought in the Andes mountain range. ARS researchers in Ithaca, New York, in collaboration with researchers in Brazil, completed phenotyping under drought and gene expression analysis in addition to fruit quality assessment and analysis of photosynthetic activity and are now identifying genes associated with these traits. In collaboration with researchers at the Boyce Thompson Institute (BTI) and in Belgium, Ithaca researchers have completed de novo sequencing of the S. lycopersicoides genome and refined high resolution molecular maps of the cross-over events defining individual population members, facilitating rapid definition of candidate genes. Ithaca researchers are exploring the degree to which whole genome transcriptome data can be influenced by genetic variation when using a single genome reference and showed that optimal gene expression data is derived from reference genomes from both parents. Indeed, expression data resulting from use of only one parental reference can be highly misleading. An informatics pipeline allowing in silico creation of reference genomes for each member of a population based on parental SNP data was developed. Understanding control of carotenoid accumulation in fruit and vegetable crops: In characterizing the nutritional carotenoid profiles of the S. lycopersicoides and other wild species IL or RIL populations, ARS researchers in Ithaca, New York, frequently identified genes contributing to carotenoid metabolism at carotenoid variation QTLs. One such gene is phytoene desaturase (PDS). It was shown that this enzyme is limiting in fruit ripening. Over expression in tomato fruit was achieved using an Arabidopsis gene that was not recognized by endogenous feed-back regulatory systems resulting in elevated carotenoid production compared to control fruit and was published. This work demonstrates that breeding for alleles with elevated PDS expression could be useful in elevating levels of nutritional carotenoids in crop plants. Researchers in Ithaca, New York, additionally advanced understanding of carotenoid accumulation by dissecting cauliflower OR gene function responsible for beta-carotene accumulation characteristic of orange cauliflower. Efforts further elucidated how OR specifically interacts with the central chloroplast division factor ARC3 to limit chromoplast division, specifically by disrupting ARC3-PARC6 interaction. They also documented a new strategy for carotenoid enrichment in crops via manipulating chromoplast number. This work was recently published. It was also shown that alteration of individual OR protein domains residing on different sides of the membrane in which OR is embedded dramatically affects OR protein stability and interaction with the key rate-limiting enzyme in the carotenoid biosynthesis pathway, phytoene synthase (PSY). This study shed new light on the contributions of OR protein domains to OR function and was also published. Finally, QTL-seq analysis along with genetic mapping identified the locus responsible for green curd color in cauliflower and localized it to a genomic region with several candidate genes. This study lays a foundation for subsequent gene isolation and provides tools for marker-assisted selection of the green curd trait in cauliflower breeding. The work was also published. Regulation of fruit ripening and nutrient quality: Efforts to characterize transcription factors regulating tomato fruit ripening have resulted in recent identification and characterization of regulators influencing specific ripening processes. One such gene, SlLOB1, is primarily responsible for influencing cell wall remodeling during fruit maturation as it regulates genes involved in cell wall synthesis, breakdown and associated textural features. A second gene functions primarily in breakdown of the locule tissue surrounding the seed prior to ripening initiation. The descriptions of these genes are largely completed and being prepared for publication. ARS researchers in Ithaca, New York, are assessing the activities of fruit and ripening-related transcription factors via various approaches that reveal sites of DNA interaction. Recent efforts have deployed DAP-seq, a high throughput in vitro adaptation of ChIP-seq, to assess DNA binding of over 100 fruit transcription factors and have also developed transcription factor-tag constructs to facilitate robust ChIP-seq of ripening regulators previously characterized by the Ithaca ARS lab and others. Twelve additional transcription factors have been identified as candidates via early ripening expression and targeted for functional analysis via CRISPR Cas-9 gene editing. Confirmed mutations have been identified for all twelve and phenotypic analysis is just beginning though at least one gene with clear ripening phenotypes when mutated in multiple independent mutant lines has been identified. Resulting gene edited lines will be used both to assess functional attributes of these genes in the fruit maturation process and will serve to address efficiency, off-targeting, and the generation of unanticipated DNA modifications resulting from gene editing to provide information on biotechnology risk of this fast emerging approach to stable genome modification. Finally, researchers in Ithaca, New York, demonstrated the ability to target gene editing to a specific tissue of a crop plant (fruit) to assess function of genes that have detrimental effects when altered in all plant tissue. This technology has high potential utility in more accurately defining gene function and was recently published. Working with collaborators from BTI, China and the U.K., researchers in Ithaca, New York, have explored the role of small non-coding RNAs in fruit ripening and have associated multiple classes of small RNA-s with the ripening process. Additionally, a small RNA specifically targeting a negative regulator of ripening ethylene synthesis was specifically shown to control levels of this transcription factor. Over-expression of the specific small RNA resulted in reduced transcription factor mRNA and elevated ethylene production. Two publications describing this work resulted.

Accomplishments
1. Vitamin A from algae. Vitamin compounds called carotenoids are vital to many life forms but are only available from plants, fungi, algae, and bacteria. In humans, vitamin A derived from carotenoids are essential for reducing the impact of chronic diseases such as cardiovascular disease, cancers, and other age-related disorders. In aquaculture and poultry industries, carotenoids are used as supplements to enhance the color of various agricultural products such as egg yolk, chicken, and fish. ARS scientists in Ithaca, New York, have created new lines of algae that increased the accumulation of several different kinds of carotenoids higher than in plants. These novel algae are useful for the production of more carotenoids.

U.S. DEPARTMENT OF AGRICULTURE

Plant, Soil and Nutrition Research: Ithaca, NY