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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

2015 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 one, ARS scientists at Charleston have collaborated with scientists at the University of Illinois Biotechnology Center and at the Boyce Thompson Institute to complete a high quality genome sequence and assembly for the heirloom watermelon cultivar ‘Charleston Gray’. The completed sequence will be made available to the public on the international Cucurbit Genomic Initiative website. In collaboration with scientists at West Virginia State University, the Charleston watermelon geneticist used the genome sequence data to conduct genotyping by sequencing to facilitate discovery of single nucleotide polymorphisms and genome-wide association mapping. This analysis identified quantitative trait loci associated with watermelon fruit quality. The watermelon genetics team has also crossed Citrullus lanatus var. citroides germplasm lines with watermelon cultivars (C. lanatus var. lanatus) to develop genetic populations segregating for Fusarium wilt race 2 resistance and for developing breeding lines resistant to Fusarium wilt race 2 that also have desirable fruit qualities. In other studies covered under objective two, watermelon breeding lines showing resistance to a zucchini yellow mosaic virus Florida strain and containing DNA markers associated with zucchini yellow mosaic virus resistance have been developed, and several lines showing superior fruit quality have also been selected. Additionally, 31 United States Plant Introductions of the desert watermelon Citrullus colocynthis L. were evaluated, and two introductions were identified as highly resistant to papaya ring-spot virus. These Plant Introductions have been used to further the development of papaya ringspot virus-resistant germplasm lines. The same 31 C. colocynthis Plant Introductions were also evaluated for resistance to whiteflies and two of them exhibiting high levels of whitefly resistance, based on low survival of adult whiteflies and a low ratio of nymphs to eggs on plants, were identified. Plants of the resistant introductions have been self-pollinated and progeny with relatively high levels of whitefly resistance have been selected for further development of germplasm lines useful in breeding programs. 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 continue to provide a means to identify the most tolerant broccoli inbreds and hybrids for possible release. Genotype by sequencing data has been obtained for a large doubled haploid population of broccoli that has been characterized for response to high temperature stress in two previous field seasons. Bioinformatics analysis of this data is currently underway. Separate studies were conducted on broccoli wherein F5 families bred for high yield and quality heads were evaluated in a replicated fall field test, and approximately 50 lines were advanced another generation for further testing in the coming 2015 fall season. An inoculated field trial was conducted in Fall 2014 that is a component of project work under objective four which is 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 fall. Additionally, a study evaluating single nucleotide polymorphism profiles of collard landraces was completed, and this work helped elucidate genetic relationships among the landraces evaluated and between collard and the other related cole crops (e.g., collard and broccoli, collard and kale, etc.). Results also indicated that genome wide association mapping is possible with the landrace collections currently under study. Relative to the subordinate project on Development of an East Coast Broccoli Industry, this project sent nine U.S. Vegetable Laboratory broccoli hybrids to the project Principle Investigator at Cornell for inclusion in the 2015 Phase One broccoli trials along with seed of four hybrids for the 2015 Phase Two trials. During the winter of 2014-15 select broccoli inbreds were cross-pollinated in the greenhouse to generate adequate seed supplies of specific hybrids for future testing. In addition, one outdoor cage was used to generate seed of two particular hybrids, and a contract seed producer was employed to produce two other hybrids in California. Year 5, Phase One trials (with 48 hybrid entries) were conducted and successfully completed in Charleston during the spring growing season as planned. Three Phase Two trials (with 15 hybrid entries) were also conducted at Charleston during March through June. Head samples from the second Phase Two trial were harvested, frozen, freeze-dried, and shipped to the University of Tennessee for nutritional testing. Two additional Phase Two trials are being initiated with plans to transplant seedlings to the field in September. All of the ARS hybrids input into the Phase One and Phase Two trials are also being evaluated for warm season adaptation by cooperating public scientists in North Carolina, New York, and Maine. For the subordinate project supported by the National Watermelon Promotion Board, project scientists used the watermelon genome sequence to identify specific gene sequences that code for ribosomal interference proteins. Eighteen gene sequences for different interference proteins and a gene sequence that encodes for a house keeping protein in watermelon were cloned from wild and cultivated watermelon accessions and are being tested in bioassays at the National Cancer Institute to determine if they have any effect on human viral activity.

1. A genetic marker that identifies watermelon with resistance to Fusarium wilt. Fusarium wilt has emerged as the most damaging disease of watermelon in the United States. Scientists on this project collaborated with the research team at a private seed company in evaluating disease response and genotyping a genetic population of watermelon segregating for resistance to Fusarium Wilt race 1, one strain of this serious disease. This research effort resulted in the identification and mapping of three single nucleotide polymorphisms and a major gene locus associated with the Fusarium Wilt race 1 resistance. The major gene identified is being used further to develop a molecular marker closely linked to the resistance gene that could be employed in public and commercial marker-assisted watermelon breeding programs aimed at incorporating wilt resistance into elite watermelon cultivars.

Review Publications
Pelc, S., Linder, C.R. 2015. Emergence timing and fitness consequences of variation in seed oil composition in Arabidopsis thaliana. Ecology and Evolution. 5(1): 164–171.
Coffey, J.L., Simmons, A.M., Shepard, B.M., Tadmor, Y., Levi, A. 2015. Potential sources of whitefly (Hemiptera: Aleyrodidae) resistance in desert watermelon (Citrullus colocynthis) germplasm. HortScience. 50:13-17.
Harrison Jr, H.F., Farnham, M.W., Jackson, D.M. 2015. Tolerance of broccoli cultivars to pre-transplanting clomazone. Crop Protection. 69:28-33.
Lambel, S., Lanini, B., Vivoda, E., Fauve, J., Wechter, W.P., Harris-Shultz, K.R., Massey, L.M., Levi, A. 2014. A major QTL associated with Fusarium oxysporum race 1 resistance identified in genetic populations derived from closely related watermelon lines using selective genotyping and genotyping-by-sequencing for SNP discovery. Theoretical and Applied Genetics. 127:2105-2115.
Stansell, Z.J., Cory, W., Couillard, D.M., Farnham, M.W. 2015. Collard land races are novel sources of glucoraphanin and other aliphatic glucosinolates. Plant Breeding. 134:350-355.
Reddy, U., Nimmakayala, P., Levi, A., Abburi, V.L., Saminathan, T., Tomasson, Y., Vajja, V.G., Reddy, R., Abburi, L., Wehner, T., Ronin, Y. 2014. High-resolution genetic map for understanding the effect of genome-wide recombination rate, selection sweep and linkage disequilibrium on nucleotide diversity in watermelon. Genes, Genomes, Genetics. 4:2219-2230.
Nimmakayala, P., Levi, A., Abburi, L., Abburi, V.L., Tomason, Y.R., Saminathan, T., Vajja, V.G., Reddy, R., Wehner, T.C., Mitchell, S.E., Reddy, U.K. 2014. Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. Biomed Central (BMC) Genomics. 15:767.
Ward, B., Smith, P., James, S., Stansell, Z.J., Farnham, M.W. 2015. Increasing plant density in eastern United States broccoli production systems to maximize marketable head yields. HortTechnology. 25:330-334.