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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

2021 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
Collaborated with researchers 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. Watermelon genome sequencing data are available to the public on the Cucurbit Genome Database (CuGenDB) website and serve as a reference for all of our genomic/genetic analysis studies. Collaborated with private seed company to generate a large genetic population 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. 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). Studies resulted in the identification a major quantitative trait locus (QTL) that contains a gene conferring Fusarium wilt resistance. 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 separate watermelon studies, we are developing breeding lines resistant to PRSV and ZYMV-Florida strain that cause serious damage to the watermelon crop. Collaborated with a seed company and screened genetic populations segregating for resistance to PRSV and ZYMV. Genomic technologies were then employed to identify a genomic region that contains the eukaryotic elongation factor (eIF4E) gene locus, which was previously determined to be tightly linked to ZYMV-resistance. Specific DNA markers were developed using sequence information and these are being useful in helping to facilitate ready movement of the virus resistance from the wild to the cultivated type of watermelon. Several plant introductions of the desert type watermelon Citrullus colocynthis, recently identified as highly resistant to PRSV, were used to develop resistant germplasm lines and genetic populations to use in genetic mapping of loci associated with resistance to PRSV. Completed the genome sequencing of the desert watermelon Citrullus colocynthis which is considered the progenitor of the cultivated type of watermelon. Identified over 880 gene sequences in the desert watermelon that may have been lost during the evolution and domestication of cultivated watermelon. Many of these lost gene sequences that exist in the desert watermelon are known to be associated with resistance to biotic and abiotic stresses. Crossing the desert and sweet types and developing genetic populations and breeding lines that should be useful for plant breeders to enhance disease resistance in modern watermelon cultivars. Completed a project of 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. Identified 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, hence improve 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” 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 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. 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. Initiated collaboration with several seed companies on the construction and development of a large multi-parent advanced intercross generation (MAGIC) population. This MAGIC population 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. Collaborated with a team at West Virginia State University (WVSU) on developing and evaluating tetraploid versus their counterpart diploid watermelon plants using advance genome sequencing technology named “Hi-C chromosome conformation capture technique”. We found that there is significantly higher number of interactions between regions from different chromosomes in the tetraploid compared with diploid plants. The difference in chromosomal interactions affect gene expression and consequently the phenotype of tetraploid versus diploid watermelon plants. The results of this study should be useful for scientists and breeders interested in developing seedless watermelon varieties. Initiated collaboration with an ARS researcher, at 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 useful for future plant breeding efforts using bioinformatics tools for the development of watermelon varieties with health promoting properties. For the broccoli portion of this project falling under Objective 2A and 2B, breeding broccoli for high temperature tolerance was continued with new tolerant selections identified and advanced another generation. Additional trials conducted in the fall allowed the project to assess performance of heat tolerant broccoli lines and hybrids under conditions more favorable to head development. Findings from those trials will help identify individuals that have the greatest commercial potential. In separate broccoli work focused on identifying vigorous inbreds that produce head yield and quality comparable to commercial hybrids, about 20 inbreds were evaluated in the field and compared to check hybrids. In work falling under Objective 3, previously screened lines of B. rapa were evaluated again for response to inoculation with the bacterium Pseudomonas cannabina pv. alisalensis (Pca) and individual resistant plants were identified. These plants were moved to a cooler, allowed to vernalize, and then placed in a greenhouse where they flowered and were self-pollinated to advance another generation. Of interest are several accessions with horticultural traits like turnip greens. Separate segregating lines of B. rapa resulting from crosses of a field-resistant line resembling Chinese cabbage and a turnip green cultivar were evaluated for resistance, and plants most like turnip greens were again selected, moved to a greenhouse, and allowed to self-pollinate. In related work, evaluated the response of 30-40 different collard lines (S3s) derived from resistant plant introductions, and these were also advanced another generation by selfing. All advanced selections will be tested an additional generation for response to inoculation with Pca. This work should lead to the identification of a blight resistant collard. Relative to the subordinate project on Development of an East Coast Broccoli Industry, eight ARS broccoli hybrids were sent to the Principal Investigator at Cornell for inclusion in the 2021 Quality trials. Additionally, seed of five hybrids were sent for on-farm yield trials in North Carolina, South Carolina, Virginia, and New York. During the winter of 2020-21, select broccoli inbreds were self-pollinated in the greenhouse to maintain adequate seed supplies for future research. Additionally, four outdoor cages were used to generate seed of four specific hybrids. All the ARS hybrids input into the Quality trials are being evaluated for warm season adaptation by cooperating public scientists in South Carolina, North Carolina, New York, and Maine. In a Cooperative Research and Development Agreement, collaborated with an industry partner to increase seed quantities of select lines of broccoli identified as producing high yields of seed with high concentration of the health-promoting compound glucoraphanin. Seed productions were conducted in the Yuma Valley in Arizona and in the Central Valley of California during winter months. Selections from segregating F4 broccoli families were made at Charleston and plants identified in the field were moved to a greenhouse and allowed to self-pollinate independently. The same F4 families were also grown in cages in Yuma during the past winter and plants with high seed yield in that environment were selected, kept in the cages, and allowed to self-pollinate absent insect pollinators. The resulting F% lines will be tested for yield potential and seed glucoraphanin content.

1. Evaluating chromosomal interactions in tetraploid and triploid versus diploid watermelon plants. Seedless watermelon plants are triploid (having three sets of chromosomes) and are the result of duplicating the number of chromosomes of a normal diploid plant (naturally having two sets of chromosomes) to have a tetraploid plant (having four sets of chromosomes). Then, the tetraploid plant is being pollinated with pollen from the diploid plant to have triploid seeds that produce plants with seedless watermelon. The tetraploid and triploid watermelon plants exhibit many phenotypic differences as compared with their diploid counterparts. Yet, there is little knowledge on the genomic and molecular events that lead to these phenotypic differences. ARS researchers in Florence, South Carolina, collaborated with a team at West Virginia State University (WVSU) on developing and evaluating tetraploid versus their counterpart diploid watermelon plants using advance genome sequencing technology named “Hi-C chromosome conformation capture technique”. We found that there is significantly higher number of interactions between regions from different chromosomes in the tetraploid compared with diploid plants. The difference in chromosomal interactions affect gene expression and consequently the phenotype of tetraploid versus diploid watermelon plants. The results of this study should be useful for scientists and breeders interested in developing seedless watermelon varieties (a feature article is highlighted in the Plant Journal

Review Publications
Vijay, J., Suhas, S., Venkata Lakshmi, A., Lopez, C., Padma, N., Levi, A., Umesh, R. 2021. Haplotype networking of GWAS hits for citrulline content indicates positive selection leading to the domestication of watermelon. Frontiers in Plant Science. 20:5392.
Garcia-Lozano, M., Natarajan, P., Levi, A., Katam, R., Nimmakayala, P., Reddy, U. 2021. Altered chromatin confirmation and transcriptional regulation in watermelon following genome doubling. Plant Journal.
Katuuramu, D.N., Wechter, W.P., Washington, M., Horry, M.I., Cutulle, M.A., Jarret, R.L., Levi, A. 2020. Phenotypic diversity for root traits andiIdentification of superior germplasm for root breeding in watermelon. HortScience. 55(8):12-72-1279.
Branham, S., Daley, J., Levi, A., Hassell, R., Wechter, W.P. 2020. QTL mapping and marker development for tolerance to sulfur phytotoxicity in melon (Cucumis melo). Frontiers in Plant Science. 11:1097-1105.
Weng, Y., Garcia-Mas, J., Levi, A., Luan, F. 2020. Editorial: translational research for cucurbit molecular breeding: traits, markers, and genes. Frontiers in Plant Science. 11. Article 615346.