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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

2016 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 falling under Objective 1, we have been developing genetic populations of Citrullus lanatus var. citroides segregating for resistance to Fusarium wilt (FW) race 2 with the goal of using these populations to identify DNA genome sequences associated with the wilt resistance. We also crossed analogous citroides lines with watermelon cultivars to develop breeding lines resistant to FW race 2 that also have the desirable fruit qualities of the cultivated type of watermelon. In collaboration with a research plant pathologist at the U.S. Vegetable Laboratory, we conducted a technique called genotyping by sequencing (GBS) to identify markers called single nucleotide polymorphisms (SNPs) that have helped us elucidate DNA sequences that are linked to genes (also called quantitative trait loci) that confer the Fusarium wilt race 2 resistance. In other related work with citroides types of watermelon, we have been utilizing specific citroides accessions collected in southern Africa to develop rootstock lines with resistance to Fusarium wilt and also to root-knot nematodes. As a result of other studies conducted under Objective 2 that are focused on viral diseases of watermelon, breeding lines that exhibit tolerance to the Florida strain of the zucchini yellow mosaic virus (ZYMV) have been developed, aided by selection for specific DNA sequences known to be associated with the ZYMV-resistance. A few of these lines also exhibit superior fruit quality. Of particular interest is the ZYMV-resistant line “USVL 370” that has good watermelon fruit quality and that has been released for public use. We recently demonstrated that several plant introductions of the desert type watermelon Citrullus lanatus var. colocynthis are highly resistant to papaya ringspot virus (PRSV), and certain of these resistant colocynthis types are being used to develop PRSV-resistant watermelon germplasm. Additionally, new genetic populations are being developed by crossing the colocynthis and cultivated watermelon types, and those populations are being used to map DNA sequences associated with PRSV-resistance. As has been done in other studies by our group, PRSV-resistant lines are being backcrossed into cultivated types of watermelon to introgress or transfer the PRSV-resistance trait into a watermelon type that exhibits desirable fruit qualities. In collaboration with a team at the Agricultural Research Organization (ARO) in Israel, we also conducted genotyping by sequencing of watermelon populations segregating for fruit quality attributes with the goal of identifying gene loci controlling flesh color, carotenoid content, and rind pattern in watermelon. In several subordinate research projects supported by the National Watermelon Research and Promotion Board, we collaborated with a team at North Carolina State University in evaluating various watermelon accessions for health promoting constituents. In one case, we identified several accessions that contain high levels of the amino acid arginine and its catabolic product citrullin, both of which have medicinal attributes, benefiting cardiovascular, metabolic and immune systems. We also identified several accessions in another study that exhibit high levels of cucurbitacin, a compound reported to confer a possible anticancer effect. In addition to the above studies, we also used the “Charleston Gray” watermelon genome sequence to identify several gene sequences that may have antiviral properties. In this work, we have been collaborating with a team at the National Cancer Institute (Frederick, Maryland) to evaluate watermelon ribosomal interference proteins (RIPs), identifying one RIP that may block the activity of human viruses. For the broccoli portion of this project falling under Objective 3, 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. Additional trials in spring and fall conducted in cooperation with a Clemson collaborator have allowed us to assess the performance of heat tolerant broccoli lines under conditions more favorable for head development. We are currently evaluating results from these trials, and those results will help determine which of our lines are most stable and have the greatest commercial potential. Genotype by sequencing was conducted for a large doubled haploid (DH) population of broccoli that was characterized for response to high temperature conditions in three previous field seasons. Bioinformatics analysis of this data has been completed, and results have identified several DNA sequences that identify genes contributing to heat tolerance. An additional inoculated field trial was conducted in the fall of 2015 that is a component of project work under Objective 4 which is focused on selecting leafy green Brassicas (mustard and turnip greens) with resistance to bacterial leaf blight disease caused by the bacterium Pseudomonas cannabina pv. alisalensis (psa). Highly resistant individuals with desirable leaf characteristics in this trial were removed from the field, allowed to self-pollinate in an outdoor cage, and harvested seed was processed for retesting in the upcoming fall. Related to this work, we are working closely with a commercial seed company, and with a number of leafy greens growers, to increase supplies of the mustard green cultivar ‘Carolina Broadleaf’ that was released by our Unit last year. Producer demand for this released mustard is very high because no existing cultivars have resistance to psa, and the disease has been very prevalent in the primary leafy green production areas in the southeast. In a separate but related study under Objective 4, a population of collard landraces, previously shown to be amenable to a process called genome-wide association mapping, was evaluated for its response to inoculation with the psa bacteria, and plans are now underway to use the association mapping technique to identify DNA sequences in collard that are linked to the psa resistance. This work could ultimately lead to the identification of a blight resistant collard.

1. Elucidating genes in broccoli that confer tolerance to high temperatures. The occurrence of relatively high temperatures during the heading stages of broccoli development can have a negative impact on head production, reducing quality of heads. Indeed, the likelihood of this environmental stress occurring in a given location or season is the single most important factor limiting where and when the crop can be grown. Breeding heat tolerant broccoli cultivars could extend the growing season, expand production areas, and increase resilience to fluctuating temperatures, but is limited by a lack of genetic knowledge about the trait. USDA scientists at Charleston, South Carolina, developed a segregating heat tolerant population of broccoli using tolerant types they developed and they evaluated lines in the population for their relative response to high temperature stress and the ability to produce good quality heads during three summer seasons in South Carolina. They used a technique called next generation sequencing in this work to identify DNA markers associated with the production of quality heads. Results of the analysis identified four gene sequences that could explain almost 60 percent of the variation in relative heat tolerance the researchers observed. The identified markers are of great interest to public and private broccoli breeders working to accelerate the development of heat tolerant broccoli cultivars.

We have been collaborating with faculty at Claflin University (Historically Black University) on efforts to advance research and applied training in Agriculture for students working at the U.S. Vegetable Laboratory. Through our collaboration, we trained a graduate student to conduct chemical analyses and they engaged those techniques to identify watermelon volatile compounds which might be associated with resistance to whiteflies.

Review Publications
Branham, S., Reba, A., Wright, S.J., Linder, C.R. 2015. Genome-wide association study of Arabidopsis thaliana identifies determinants of natural variation in seed oil composition. Journal of Heredity. doi:10.1093/jhered/esv100.
Branham, S., Couillard, D.M., Stansell, Z.J., Farnham, M.W. 2015. Genetic diversity and population structure of collard landraces and their relationship to other Brassica oleracea crops. The Plant Genome. 8(3):1-11.
Branham, S., Reba, A., Wright, S.J., Linder, C.R. 2016. Genome-wide association study in arabidopsis thaliana of natural variation in seed oil melting point, a widespread adaptive trait in plants. Journal of Heredity. doi: 10.1093/jhered/esw008.
Farnham, M.W., Branham, S. 2016. Glucoraphanin and other glucosinolates in heads of broccoli cultivars. Broccoli: Cultivation, Nutritional Properties and Effects on Health. B.H.J. Juurlink (Ed.). Nova Science Publishers. New York, NY. 342 p.
Coffey, J.L., Simmons, A.M., Shepard, B.M., Levi, A. 2016. A vertical Y-tube is a valuable tool for assessing whitefly preference, yielding well-defined results among attractive versus poor host plants. Journal of Agricultural and Urban Entomology. 32(1):7-12.
Kim, H., Han, D., Kang, J., Choi, Y., Levi, A., Park, Y. 2015. Sequence-characterized amplified polymorphism markers for selecting rind stripe pattern in watermelon (Citrullus lanatus var. lanatus). Journal of Horticulture, Environment and Biotechnology. 56:341-349.
Thies, J.A., Ariss, J., Kousik, C.S., Hassell, R.L., Levi, A. 2016. Resistance to Southern Root-knot Nematode (Meloidogyne incognita) in Wild Watermelon (Citrullus lanatus var. citroides) Populations. Journal of Nematology. 48:14-19.
Paris, H., Faigenboim, A., Reddy, U., Donahoo, R.S., Levi, A. 2015. Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers. Genetic Resources and Crop Evolution. 62:1095-1111.
Levi, A., Harris-Shultz, K.R., Ling, K. 2016. USVL-370, A zucchini yellow mosaic virus resistant watermelon breeding line. HortScience. 51:107-109.
Levi, A., Coffey, J., Massey, L.M., Guner, N., Oren, E., Tadmor, Y., Ling, K. 2016. Resistance to papaya ringspot virus-watermelon strain (PRSV-W) in the desert watermelon Citrullus colocynthis. HortScience. 51:4-7.