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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

2023 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
This is the final report for this project. See new project 6080-21000-020-000D for additional information. This project has been focusing on genetic research and breeding of watermelon, broccoli, and leafy green Brassicas for resistance to major diseases, tolerance to environmental stress, and enhanced quality or nutritional attributes to help stakeholders, including seed companies, growers, producers, and consumers. Significant progress has been made in identifying and developing new resources of resistance and incorporating disease resistance gene loci into watermelon cultivars. In addition, significant progress has been made in identifying and utilizing gene loci conferring heat tolerance and in developing and releasing heat tolerant broccoli lines. The project with 2 SY (2018-2019) and 1 SY (2020-2022) successfully published 38 peer reviewed journal manuscripts related to watermelon or cucurbit crops, 8 peer reviewed journal manuscripts related to broccoli, and released two heat tolerant broccoli breeding lines (USVL156 and USVL160 inbred lines) adapted to hot summer environments of the eastern United States. USDA ARS scientists in Charleston, SC released a zucchini yellow mosaic virus (ZYMV) and papaya ringspot virus (PRSV)-resistant watermelon line “USVL-380. They also co-released a plant variety protected (PVP)- rootstock line “Carolina Strongback” having resistance to fusarium wilt and root-knot nematode and that is useful for grafting watermelon cultivars. Disease resistant watermelon and heat tolerant lines were provided to several national and international seed companies and universities through material transfer agreements (MTA). We have conducted genomic and genetic analyses studies, and in collaboration with the bioinformatics team at the Boyce Thompson Institute (Cornell University) we sequenced and assembled the genome of the principle American watermelon cultivar “Charleston Gray” and re-sequenced 1300 United States Plant Introductions (PIs) of the USDA-ARS watermelon (Citrullus spp.) germplasm collection. Using advanced genomic approaches, we were able to identify gene loci associated with cold tolerance, downy mildew, and powdery mildew resistance in watermelon. We conducted genetic analysis studies and identified quantitative trait loci (QTL) associated with resistance to Fusarium wilt races 1 and 2 of watermelon, papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) of watermelon. We have developed and released a self-compatible green sprouting broccoli cultivar ‘HiTest’ that yields seed with high levels of the phytonutrient Glucoraphanin which is known to have health benefits. All these work and new research projects are being continued in the new five-year project plan (6080-22000-020-000D; 2023-2028) to provide stakeholders with genomic and genetic tools and germplasm useful for enhancing biotic and abiotic stress tolerance in vegetable crops. Analyzed watermelon genetic populations 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 genetic populations were analyzed using the genotyping by sequencing (GPS) technology 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). The genetic analysis identified several major quantitative trait loci (QTL) conferring resistance to Fusarium wilt of watermelon. DNA markers associated with these resistance gene loci have been developed and validated and proved useful in our breeding program and to plant breeders aiming to incorporate FW-resistance into elite watermelon cultivars. A gene locus that confers resistance to PRSV and ZYMV was identified. 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. Employed genomic technologies to identify the eukaryotic elongation factor (eIF4E) gene locus, which was previously determined to be tightly linked to ZYMV-resistance. Developed DNA markers useful for incorporating the gene locus conferring virus resistance from the wild type into elite watermelon cultivars. In a separate study, identified two ribosomal inactivating proteins (RIPs) highly expressed in response to ZYMV-infection. These two RIP genes are likely to play an important role in resistance to potyviruses. This is in addition to the eIF4E gene locus, and they could be useful in our breeding programs aimed to enhance resistance in watermelon cultivars. 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) resistant to various diseases of watermelon and representing most Citrullus spp. (including C. amarus, C. colocynthis, C. lanatus and C. mucosospermus). In collaboration with a bioinformatics team at the Boyce Thompson Institute (Cornell University), we have been constructing a Pan-Genome for watermelon and identified a large number of gene sequences that exist in watermelon accessions collected in the wild but not in the genome of watermelon cultivars. These sequences are designated as dispensable and many of them are associated with resistance to biotic and/or abiotic stress and might have been lost during the many years of domestication of 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. Structural genomic variations (including insertions and deletions), represent a major source of genetic diversity and are related to numerous agronomic traits and evolutionary events in crop plants. Comprehensive identification and characterization of such structural variations in melon have been hindered by the lack of a high-quality pan-genome. In collaboration with the ARS-Southeast Bioinformatics Team, we have utilized an advance genomic procedure known as “Graph-based genomic alignment approach” to construct a high-resolution reference genome representing of all polymorphic occurrences in the melon genome. The graph-based reference genome improves genetic information density relative to prior sequence assembly studies and should be highly useful in breeding programs and in elucidating gene loci associated with disease resistance and fruit quality in melon. In collaboration with an ARS plant pathologist at the U.S. Vegetable Laboratory, Charleston and with scientists at Cornell University and North Carolina 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. Identified two major gene loci associated with PM-resistance and developed and validated molecular markers for two loci. These molecular markers should be useful for increasing PM-resistance in elite watermelon cultivars. Through our ongoing CucCAP project (6080-21000-019-11R) collaborating with researchers 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. 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 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, successfully mapped 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. As part of the CucCAP project objectives, completed the first step in the construction and development of a large multi-parent advanced intercross generation (MAGIC) population for watermelon and have been collaborating with seed companies in the development of the MAGIC-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. Through the CucCAP project “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops” we have been collaborating with researchers at West Virginia State University (WVSU) on a genetic mapping study to identify quantitative trait loci (QTL) associated with resistance to gummy stem blight (GSB) which is considered a major soilborne disease of watermelon. Using a recombinant inbred line population derived from crosses between GSB-resistant and susceptible parental lines, we were able to identify several QTL and candidate gene loci associated with GSB-resistance. The results and DNA markers developed through this study should be useful in breeding programs aiming to develop watermelon varieties with resistance to GSB.

1. Sources of disease resistance in watermelon. Cucurbit downy mildew (CDM) is a serious threat to the production of cucumber, cantaloupes, and watermelon in the U.S as well as in other cucurbit growing areas around the world. New sources of resistance and molecular markers are needed to control the spread of CDM pathogen in watermelon. ARS researchers in Charleston, South Carolina, surveyed USDA plant introductions (PIs) and identified several sources that are resistant to CDM. In addition, we identified gene loci associated with CDM-resistance in watermelon. The CDM-resistant PIs and molecular markers for gene loci associated with resistance will be useful in breeding programs aimed to enhance CDM-resistance in watermelon cultivars.

Review Publications
Vaughn, J.N., Branham, S.E., Abernathy, B.L., Hulse-Kemp, A.M., Rivers, A.R., Levi, A., Wechter, W.P. 2022. Graph-based pangenomics maximizes genotyping density and reveals structural impacts on fungal resistance in melon. Nature Genetics.
Gursewak, S., Ward, B., Levi, A., Cutulle, M. 2022. Weed management by summer cover crops, solarization and anaerobic soil disinfestation in plasticulture. HortScience. 12(11):2754.
Reddy, U.K., Natarajan, P., Lakshmi Abburi, V., Tomason, Y., Levi, A., Nimmakayala, P. 2022. What makes a giant fruit? Assembling a genomic toolkit underlying various fruit traits of the mammoth group of Cucurbita maxima. Frontiers in Plant Science.
Waldo, B.D., Branham, S.E., Levi, A., Wechter, W.P., Rutter, W.B. 2022. Distinct genomic loci underlie quantitative resistance to Meloidogyne enterolobii galling and reproduction in Citrullus amarus. Plant Disease. 107:2126-2132.