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ARS Home » Southeast Area » Charleston, South Carolina » Vegetable Research » Research » Research Project #425105

Research Project: Genetic Enhancement of Watermelon, Broccoli, and Leafy Brassicas for Economically Important Traits

Location: Vegetable Research

2014 Annual Report

1. Devise sequence-based markers to accelerate the transfer of new sources of resistance to Fusarium wilt and potyviruses from wild to cultivated watermelon types. 1.A. Utilize watermelon genome sequence to develop a single nucleotide polymorphism (SNP)-based linkage map for the citron watermelon, identifying markers associated with Fusarium wilt (FW) and Papaya ring spot virus (PRSV) resistances. 1.B. Develop a SNP-based genetic linkage map for the cultivated type watermelon (C. lanatus var. lanatus) which includes markers associated with PRSV resistance and also fruit attributes. 2. Develop and release watermelon germplasm with improved resistance to Fusarium wilt and potyviruses combined with improved phytonutrient content. 2.A. Develop and release watermelon germplasm exhibiting FW, race 2 resistance from the wild “citron” combined with attributes (e.g. presence of lycopene) of cultivated watermelon. 2.B. Develop and release watermelon germplasm exhibiting resistance to Zucchini yellow mosaic virus (ZYMV) combined with attributes of cultivated watermelon. 3. Breed and release broccoli lines with enhanced tolerance to high temperature stress by incorporating additional, new tolerance genes, and develop broccoli with divergent levels of health promoting compounds. 3.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. 3.B. Develop genetically similar broccoli lines with divergent levels of glucoraphanin useful for studying the human health promoting effects of this vegetable. 4. Exploit genotypic and phenotypic diversity in leafy green Brassica germplasm to develop lines with resistance to bacterial leaf disease and enhanced levels of health promoting compounds. 4.A. Develop an inbred line of leafy mustard green (B. juncea) with resistance to Pseudomonas cannabina pv. alisalensis (Pca) and improved horticultural phenotype, and a line of B. rapa with resistance to Pca. 4.B. Examine genotypic and phenotypic diversity in a unique collection of collard landraces collected from southern seed savers, and identify useful sources of disease resistance and phytonutrient profiles in this germplasm.

Select parental lines of watermelon, broccoli or leafy green Brassicas based on phenotypic expression of resistance, tolerance or quality traits under study. Use the selected parent lines to construct conventional (i.e., F2, BC1, recombinant inbred) and doubled haploid (for broccoli only) populations segregating for the traits of interest, and then employ those populations in studies to determine mode of inheritance of each character or to select superior lines. Utilize PCR-markers and other genomic technologies, such as genotype by sequencing, to identify sequences linked to the studied characters and to locate controlling genes on linkage maps. Use particular markers (e.g., SSRs, SNPs, or SCARs) closely associated with traits of interest to develop tools for marker-assisted selection. Based on knowledge gained through the above studies, devise breeding strategies, and applications of marker technologies to use in the further development of horticulturally-enhanced lines or hybrids that express resistances and other traits of interest and that also produce high quality vegetables. Make enhanced lines available through public releases or commercial licensing. Continue ongoing searches for new resistances or tolerances among watermelon and vegetable Brassica accessions from the U.S. Plant Introduction and other collections.

Progress Report
For the watermelon portion of this project and research relative to objective 1, the genome of the watermelon heirloom cultivar ‘Charleston Gray’ has been sequenced and assembled with 93.5% coverage of the plant’s genome in collaboration with scientists at the University of Illinois Biotechnology Center and the Boyce Thompson Institute (Ithaca, New York). Using the watermelon genome sequence data, genotyping by sequencing (GBS) was conducted to elucidate genetic markers called single nucleotide polymorphisms (SNPs). These markers were used to study F2 and F3 genetic populations segregating for Fusarium wilt (FW) race 1 resistance. Three SNPs and a major quantitative trait locus (QTL) were identified as associated with the race 1 resistance. Crosses between lines of one wild watermelon relative (Citroides type) were crossed with watermelon cultivars to develop genetic populations segregating for FW race 2 resistance, and to initiate development of breeding lines that are resistant to this damaging FW race and that also have desirable fruit qualities. In other watermelon studies covered under objective 2, breeding lines showing tolerance to a Florida strain of zucchini yellow mosaic virus (ZYMV) and containing DNA markers previously shown to be associated with ZYMV resistance have been developed, and several of these lines showing superior fruit quality have been identified. For the broccoli portion of this project falling under objective three, an additional cycle of breeding broccoli for tolerance to high temperature stress was completed, and new tolerant selections were identified and advanced another generation. Replicated trials in the summer at Charleston are helping to identify the most tolerant broccoli inbreds and hybrids for possible germplasm release. A large replicated field trial of a doubled haploid (DH) population (160 individuals) of broccoli that segregates for tolerance to high temperature stress has been conducted; results continue to indicate quantitative inheritance of this trait. This DH population is currently being prepared for genotyping by sequencing in order to specifically identify gene sequences associated with the tolerance trait. In separate work on broccoli, F4 families bred for high yield and quality heads were evaluated in fall nurseries and the best lines (approx. 50) were selected and advanced to the next generation for future testing. An inoculated field trial was conducted in the autumn relative to project work under objective four focused on selecting leafy green Brassicas (i.e., mustard and turnip greens) with resistance to bacterial leaf blight disease. Highly resistant individuals with desirable leaf characteristics were removed from the field, allowed to self-pollinate in an outdoor cage, and harvested seed was processed for retesting in the upcoming autumn. Lastly, a study evaluating glucosinolate profiles of collard landraces was completed, and this work identified several specific landraces that contain high levels of glucoraphanin, a glucosinolate previously thought to be abundant only in broccoli and that has high potential as a health-promoting compound. Relative to the subordinate project on Development of an East Coast Broccoli Industry, this project sent nine U.S. Vegetable Laboratory (USVL) broccoli hybrids to the project PI at Cornell for inclusion in the 2014 Phase 1 broccoli trials along with seed of four hybrids for the 2014 Phase 2 trials. During the winter of 2013-14 select broccoli inbreds were cross-pollinated in the greenhouse to generate adequate seed supplies of specific hybrids for future testing. In addition, two outdoor cages were used to generate seed of four particular hybrids. Year 4, Phase 1 trials (with 39 hybrid entries) were conducted and successfully completed in Charleston, South Carolina during the spring growing season as planned. Three Phase Two trials (with 15 hybrid entries) were also conducted at Charleston, South Carolina during March through June. Head samples from the second Phase 2 trial were harvested, frozen, freeze-dried, and shipped to Knoxville, Tennessee for nutritional testing. Two additional Phase 2 trials are being initiated with plans to transplant seedlings to the field in September. All of the ARS hybrids input into the Phase 1 and Phase 2 trials are also being evaluated for warm season adaptation by cooperating public scientists in North Carolina, New York, and Maine. In subordinate project work in conjunction with the National Watermelon Association, U. S. Plant Introductions (PIs) of a wild desert watermelon (Colocynthis type) were evaluated, and two PIs were identified as highly resistant to papaya ring-spot virus (PRSV). Selections from these PIs have been made and are being used to develop PRSV-resistant lines. The selections are also being used as parental lines for developing populations segregating for PRSV resistance. For the subordinate project supported by the National Watermelon Promotion Board, we used the watermelon genome sequence to identify specific gene sequences that code for ribosomal interference proteins (RIP). Eighteen such RIP gene sequences were cloned from wild and cultivated watermelon accessions and shipped to the National Cancer Institute. These clones are being investigated to determine if they have any effect on human viral activity.

1. Release of elite broccoli inbreds. Nearly all new cultivated varieties of broccoli are hybrids formed by crossing two inbred lines. The actual breeding and selection of inbred broccoli lines is a time-consuming, but necessary, initial part of the entire process that ultimately culminates in the development of new hybrid varieties for use in commercial production. This project at the U.S. Vegetable Laboratory in Charleston, South Carolina, has been developing a diverse array of inbred lines that can be useful in making hybrids. Repeated tests at the Vegetable Laboratory identified two particular inbreds, designated USVL048 and USVL131, which were developed at the Charleston, South Carolina ARS location. These two inbreds make excellent broccoli hybrids when they are crossed to other inbreds. In general, these two new inbreds produce hybrids that have very good head quality attributes and that may be useful as commercial varieties. Broccoli breeders at public universities and with private industry are interested in obtaining seed of USVL048 and USVL131, and have found these inbred lines to be valuable additions to their respective breeding programs aimed at broccoli improvement.

Review Publications
Farnham, M.W., Grusak, M.A. 2014. Assessing nutritional changes in a vegetable over time: issues and considerations. HortScience. 49:128-132.
Levi, A., Thies, J.A., Wechter, W.P., Farnham, M.W., Weng, Y., Hassel, R. 2013. USVL-360, a novel watermelon tetraploid germplasm line. HortScience. 49:354-357.
Davis, A.R., Webber III, C.L., Liu, W., Perkins-Veazie, P., Levi, A., King, S. 2013. Watermelon quality traits as affected by ploidy. HortScience. 48:1113-1118.
Tomason, Y., Nimmakayala, P., Levi, A., Reddy, U.K. 2014. Map-based molecular diversity, linkage disequilibrium and association mapping of fruit traits in melon. Molecular Breeding. 31:829-841.
Farnham, M.W. 2013. Doubled haploid inbred lines USVL048 and USVL131 of heading broccoli. HortScience. 48:907-908.