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ARS Home » Southeast Area » Charleston, South Carolina » Vegetable Research » Research » Research Project #434575

Research Project: Genomics and Genetic Improvement of Disease Resistance and Horticultural Characteristics of Watermelon, Broccoli, and Leafy Brassicas

Location: Vegetable Research

2022 Annual Report

Objective 1. Develop genomic tools and use them to develop and release watermelon germplasm with improved disease resistance, combined with desirable fruit quality and other consumer- and commercially-relevant horticultural traits. Sub-objective 1.A. Utilize an identified major quantitative trait locus (QTL) for Fusarium wilt Race 2 resistance to develop sequence-based markers as selection tools to aid the incorporation of resistance into enhanced watermelon germplasm with desirable fruit characteristics. Sub-objective 1.B. Utilize the watermelon genome sequence to develop a single nucleotide polymorphism (SNP)-based linkage map for the desert watermelon (Citrullus colocynthis) and identify markers associated with resistance to Papaya ring spot virus (PRSV). Sub-objective 1.C. Develop and release watermelon germplasm with improved disease resistance from a wild watermelon type combined with improved fruit characteristics of cultivated types. Objective 2. Develop and release broccoli germplasm with improved adaptation to high temperature environments and other commercially- and consumer-relevant horticultural traits. Sub-objective 2.A. Breed and release broccoli lines with enhanced tolerance to high temperature by exploiting additional, new tolerance alleles, and identify genomic sequences associated with the tolerant phenotype. Sub-objective 2.B. Determine if elite broccoli inbreds that are vigorous and highly self-compatible can produce head yield and quality comparable to that of commercial hybrid broccoli cultivars. Objective 3. Utilize genetic diversity in leafy green Brassicas (B.) to develop germplasm with improved commercially- and consumer-relevant traits. Sub-objective 3.A. Determine mode of inheritance of resistance to Pseudomonas cannabina pv. alisalensis (Pca) in a B. rapa accession with turnip-like leaves. Sub-objective 3.B. Exploit phenotypic diversity in a unique collection of collard landraces collected from southern seed savers to develop a B. oleracea collard with resistance to Pca and another collard that expresses relatively high levels of the glucosinolate glucoraphanin.

Parental lines of watermelon, broccoli or leafy green Brassicas will be selected based on phenotypic expression of resistance, tolerance or quality traits under study. The selected parental lines will then be utilized to construct conventional (i.e., F2, BC1, recombinant inbred) and doubled haploid (for broccoli only) populations segregating for the traits of interest. These populations will in turn be used in studies to determine mode of inheritance of each character or to select new, more superior lines. Modern techniques like genotyping by sequencing or quantitative trait locus (QTL) seq will be employed to identify DNA sequences associated with the traits of interest and to locate controlling genes on genetic linkage maps. Key DNA sequences will be used to develop strategic markers, e.g. kompetitive allele specific primer (KASP) markers, that are closely linked to the traits under study and that can be used in marker-assisted selection strategies. Knowledge gained in the above studies will be applied in developing improved breeding approaches and in fine-tuning marker-assisted methods to use in the further development of enhanced horticultural lines or hybrids that express improved resistances or tolerances and other traits of interest and that also produce high quality vegetable products. The improved plant germplasm will be made available through public releases or commercial licensing. Ongoing searches for new resistances or tolerances among watermelon and vegetable Brassica accessions from the U.S. Plant Introduction and other collections will also be conducted.

Progress Report
For Objectives 1A and 1B, we have collaborated with scientists at Cornell University on sequencing, developing, and utilizing the ‘Charleston Gray’ genome and the genomes of a large number of United States Plant Introductions (PIs) representing most Citrullus spp. (including C. amarus, C. colocynthis, C. lanatus and C. mucosospermus) and resistant to various diseases of watermelon. In collaboration with the Cornell team, we have been constructing a Pan-Genome for watermelon and have identified a large number of gene sequences that exist in the wild type watermelons and have been lost during the many years of evolution and domestication of the sweet desert watermelon. The watermelon genome sequencing data are available to the public on the Cucurbit Genome Database (CuGenDB) website and serve as a reference for all our genomic/genetic analysis studies. We have developed genetic populations for watermelon segregating for resistance to Fusarium wilt (FW; races 1 and 2), which is considered the most destructive disease of watermelon in the USA and throughout the world. The developed populations were analyzed using advanced DNA technologies and were evaluated for resistance to Fusarium wilt race 2, papaya ring spot virus (PRSV) and zucchini yellow mosaic virus (ZYMV)-Florida Strain (significant diseases of watermelon). A genetic mapping study resulted in the identification of a major quantitative trait locus (QTL) conferring resistance to Fusarium wilt of watermelon. Also in this study, a gene locus that confers resistance to PRSV and ZYMV was identified. DNA markers associated with the resistance gene loci have been developed and proved useful in our breeding program and to plant breeders working to incorporate the resistance gene loci from the wild into elite watermelon cultivars. In a separate watermelon study relative to Objective 1C, we have developed breeding lines resistant to PRSV and ZYMV-Florida strain that cause serious damage to the watermelon crop. In collaboration with a seed company, we have screened genetic populations segregating for resistance to PRSV and ZYMV. We employed genomic technologies to identify the eukaryotic elongation factor (eIF4E) gene locus, which was previously determined to be tightly linked to ZYMV-resistance. We developed DNA markers useful for incorporating the gene locus conferring virus resistance from the wild type into elite watermelon cultivars. In a separate study, we identified two ribosomal inactivating proteins (RIPs) that are highly expressed in response to viral infection. These two RIP genes likely to play an important role in resistance to potyviruses in addition to eIF4E gene locus and could be used in our breeding programs aimed to enhance resistance in watermelon cultivars. We completed screening and evaluating root systems of 400 watermelon accessions and determined that the root system of many cultivars is small and deficient in secondary fibrous roots. We identified wild watermelon accessions with extensive root system or accessions having a high number of secondary fibrous roots that could be useful to improve the root system of watermelon cultivars. The phenotypic and genotyping data developed are currently being used in a genome wide association study (GWAS) for the identification and development of molecular marker tightly linked to gene loci controlling root traits and can be useful for improving root systems in watermelon cultivars, hence improving their tolerance to soil-borne diseases and/or abiotic stresses like drought. Through our ongoing CucCAP project “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops” we have been collaborating with scientists at Cornell University, Michigan State, North Carolina State University (NCSU), and University of Illinois on sequencing the watermelon genome and on developing a Pan-Genome for watermelon that includes all watermelon (Citrullus spp.) species and subspecies. We sequenced the genome of 1,365 wild watermelon accessions and in collaboration with seed companies we have been developing a core collection which includes 384 selected PIs useful for screening for disease or pest resistance. In collaboration with the CucCAP team at NCSU we have been using a technology named “Resistance gene enrichment sequencing (RenSeq)” and were able to successfully reannotate and map gene sequences associated with resistance gene family. Also, we were able to successfully map quantitative trait loci (QTL) associated to major diseases of watermelon, including Fusarium wilt race 2, PRSV, ZYMV and bacterial fruit blotch. The data from these studies are available on the Cucurbit Genome Database (CuGenDB) website and are being used by seed companies for improving disease resistance in elite watermelon cultivars. Through the CucCAP project and in collaboration with an ARS plant pathologist at the U.S. Vegetable Laboratory, and scientists at Cornell University, North Carolina State University and Michigan State University, we employed a technology known as extreme-phenotype genome-wide association study (XP-GWAS) to identify gene loci associated with resistance to powdery mildew (PM) of watermelon. Using the XP-GWAS technology we identified two major gene loci associated with PM-resistance. We developed and validated molecular markers for these two loci that should be useful for seed company plant breeders in enhancing PM-resistance in elite watermelon cultivars. As part of the CucCAP project objectives, we have completed the first step in the construction and development of a large multi-parent advanced intercross generation (MAGIC) population for watermelon and have given seeds to seed companies to proceed in the development of recombinant inbred lines (RILs). This MAGIC population (developed in collaboration with six seed companies) will be a robust germplasm source with diverse allelic combinations, useful to watermelon breeders and provide an opportunity for exploring the Citrullus spp. genome interactions, track introgressions and chromosomal recombination as well as conduction of fine genetic mapping (6080-21000-019-11R). We collaborated with a team at West Virginia State University (WVSU) on a genome-wide association study (GWAS) to identify gene loci playing a role in the metabolism of watermelon seed-bound amino acids. Through this collaborative study, we identified several candidate genes involved in metabolic pathways associated with amino acid metabolism. The results of this study provide a platform for exploring potential gene loci involved in seed-bound amino acids metabolism, useful in the development of watermelon varieties with superior seed nutritional values. The project on developing an eastern broccoli Industry through cultivar development, economically and environmentally sustainable production and delivery was completed (6080-21000-019-09R). The project on testing combining ability of ARS broccoli inbred lines in hybrid combinations was completed prior to the scientist retiring (0000062435).

1. Developed a catalog for all known phytochemicals occurring in watermelon. An ARS researcher in Charlston, South Carolina, initiated collaboration with the Nutrition and Genomics Laboratory, JM-USDA Human Nutrition Research Center on Aging at Tufts University, on identifying and cataloging all known natural compounds of watermelon. Through this collaboration we were able to successfully identify and develop a catalog of 1557 small molecules (phytochemicals) present in watermelon and identify a large group of antioxidant molecules and a group of molecules with diuretic effect. The data of this collaborative study are a useful platform for future plant breeding efforts using bioinformatics tools for the development of watermelon varieties with health promoting properties. The functional bioactive compounds identified in watermelon possess enormous structural and chemical diversity and might be an invaluable source for future studies aiming for discovery of therapeutic drugs. This study was recently published in Frontiers in Nutrition.

Review Publications
Joshi, V., Nimmakayala, P., Song, Q., Lakshmi, V.A., Natarajan, P., Levi, A., Crosby, K., Reddy, U. 2021. Genome-wide association study and population structure analysis of seed-bound amino acids and total protein in watermelon. Plant Science.
Chanda, B., Wu, S., Fei, Z., Ling, K., Levi, A. 2022. Elevated expression of ribosome-inactivating protein (RIP) genes in potyvirus-resistant watermelon in response to viral infection. Canadian Journal of Plant Pathology. 44(4):615-625.
Katuuramu, D.N., Branham, S.E., Levi, A., Wechter, W.P. 2022. Genome-wide association analysis of resistance to Pseudoperonospora cubensis in citron watermelon. Plant Disease. 106:1952-1958.