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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

2018 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
This is the final report for this project which has been replaced by project 6080-21000-019-00D, "Genomics and Genetic Improvement of Disease Resistance and Horticultural Characteristics of Watermelon, Broccoli, and Leafy Brassicas." For the watermelon portion of this project falling under Objective 1, this project collaborated with a Research Plant Pathologist at the Charleston, South Carolina location to select and develop watermelon germplasm lines USVL-246FR and USVL-252FR exhibiting strong resistance to Fusarium Wilt (FW). These lines have been used in breeding programs of several seed companies working to incorporate FW resistance into elite watermelon lines. In collaboration with an industry partner, we evaluated an F2:3 genetic population of Citrullus lanatus for FW race 1 resistance and used genotyping by sequencing (GBS) to construct a single nucleotide polymorphism (SNP)-based genetic map and to identify a major quantitative trait locus (QTL) and four minor QTL associated with the resistance. With the same population, we used QTL-seq analysis and sequenced genomic DNA from a subset of the most resistant lines versus a subset of the most susceptible. This analysis identified a cluster of SNPs highly associated with FW race 1 resistance. In other collaborative research at the U.S, Vegetable Laboratory (USVL), we constructed genetic populations (F1, F2, BC1R, BC1S, and F3) segregating for FW race 2. Using GBS, we were able to construct a SNP-based genetic linkage map and identify a major QTL associated with resistance to FW race 2 derived from USVL-246FR. In other watermelon work, we identified a new source of resistance to Papaya Ring Spot Virus (PRSV) in the desert watermelon C. colocynthis and have been making advances in developing PRSV-resistant germplasm lines that will in turn be used to develop genetic populations for future studies. This project also facilitated the sequencing and assembly of the watermelon heirloom cultivar “Charleston Gray” genome in collaboration with scientists at the University of Illinois, Biotechnology Center, and the Boyce Thompson Institute at Cornell University. The genomic data (DNA sequence) from this project has been published on the International Cucurbit Genomic Initiative website and is being used extensively by public-sector and private-sector scientists throughout the world to elucidate DNA sequences controlling disease resistance and numerous other economically-important horticultural traits of watermelon. In separate watermelon studies relative to Objective 2, we used DNA-based molecular markers developed in previous studies to facilitate breeding of watermelon lines USVL-370 and USVL-380 with resistance to the Zucchini yellow mosaic virus (ZYMV). These new lines are homozygous for the recessive eukaryotic elongation factor eIF4E allele associated with ZYMV resistance originally identified in U.S. PI 595203. The project is also making some progress incorporating FW resistance from USVL-252 FR into cultivated watermelon backgrounds, with current BC3F2 populations now exhibiting FW race 2 resistance. These populations will be further advanced with the aim of developing elite watermelon germplasm with FW race 2 resistance combined with desirable fruit characteristics. Relative to Objective 3 of this project, work focused on broccoli, cycles of hybridization and inbreeding followed by selection in summer broccoli trials which resulted in the development of broccoli germplasm increasingly adapted to high temperature environments like those that occur in Charleston, South Carolina during late spring and summer. The project is currently considering the formal release of a hot season-tolerant inbred to occur under the auspices of the replacement project. In addition, phenotypic data is being collected for several candidate hybrids also tolerant of hot conditions that may be used in submitting a Plant Variety Protection (PVP) Certificate application if approved to do so. Wide-scale testing of project hybrids along the eastern seaboard has been facilitated by this project’s active participation in a NIFA-funded Specialty Crops Research Initiative Comprehensive Agricultural Project entitled “Developing an eastern broccoli industry through cultivar development, economically and environmentally sustainable production and delivery”. In research most central to this objective, a population of about 160 doubled haploid (DH) broccoli lines segregating for high temperature response was evaluated over three summers in replicated field trials. The resulting phenotypic response data was used in combination with a genotyping by sequencing approach to identify QTLs associated with heat tolerance. Results from this study confirmed the quantitative nature of the high temperature tolerance, identifying five QTL and one possible epistatic effect that explained 62% of the variation for the tolerance trait in the test population. At this time, there is no clear indication that the USVL efforts to enhance high temperature stress tolerance have reached a plateau. On the contrary, there are many indications that further advances can be made, primarily due to the introgression of new factors from additional and different broccoli backgrounds. Moreover, a second segregating DH population evaluated more recently has helped elucidate two or three additional QTLs not previously identified. Broccoli research on other important traits (e.g., glossy leaves), expressed by DH lines released by the project, has helped elucidate genetic control and important gene sequences associated with these characters. Relatively recent inbred line releases by the project include one inbred shown to exhibit a relatively high concentration of glucoraphanin as well as good combining ability for levels of this health-promoting constituent. A critical accomplishment of this project’s ongoing efforts to control bacterial leaf blight on vegetable Brassicas in the last few years, was the release of a new cultivar of mustard greens (Brassica juncea) named ‘Carolina Broadleaf’. This project led the breeding efforts in developing this cultivar that exhibits high levels of resistance to Pseudomonas cannabina pv. alisalensis (Pca) and that is the only known leafy green Brassica cultivar with resistance to this pathogen. Advances have also been made in developing resistant B. rapa lines originating from the vegetable B. rapa plant introduction designated G30499. These plant introduction-derived lines have been selfed several generations and now appear uniform and stable, exhibiting what appears to be “field resistance”. However, these lines look more like Chinese cabbage, and the desired phenotype is more akin to a turnip green. Efforts to transfer the resistance into a turnip green phenotype are ongoing. Work of this project focused on collard greens has better characterized a group of collard landraces previously collected by this project and deposited into the USDA Plant Germplasm System. A 60K Brassica SNP BeadChip array with 52,157 SNPs was used to clarify the relationships of collard to other B. oleracea crops, to evaluate genetic diversity and population structure, and to assess the potential for employing genome-wide association mapping to identify sequences associated with traits segregating in the collard population. Additional studies have verified that the collard collection contains unique germplasm that might be used to develop value-added collards that exhibit specific attributes like elevated levels of glucoraphanin. Current studies also indicate that particular accessions from the collard collection exhibit significant resistance to Pca and might be used to develop a blight resistant collard.

1. Identification of additional genes in broccoli that confer tolerance to high temperatures. High temperatures occurring during the early stages of broccoli head development are usually detrimental to the harvested vegetable, causing distorted, misshapen heads that are not marketable. Indeed, the likelihood of heat damage to heads occurring in a given location or season is the most important factor limiting where and when broccoli is grown. Breeding heat tolerant broccoli cultivars could extend the growing season, expand production areas, and increase resilience to fluctuating temperatures, but this effort has been limited by a lack of genetic knowledge about the trait. ARS researchers at Charleston, South Carolina, compared the DNA make-up of lines from the same population that respond very differently to high temperatures, with one group quite tolerant of the heat and able to produce good quality heads at high temperatures, and a second group not tolerant of heat at all, only producing very poor quality heads at high temperatures. Using a new gene detection method, the scientists were able to identify two new spots on broccoli chromosomes that appear to pinpoint genes that confer heat tolerance in this important vegetable. These identified genes are of great interest to public and private broccoli breeders working to accelerate the development of heat tolerant broccoli cultivars.

2. Development of DNA-based markers that can be used to aid the breeding of watermelon with resistance to Fusarium wilt. Fusarium wilt is a major disease of watermelon which can cause significant losses to growers throughout the United States. The causal agent of this disease is the soil-borne fungus named Fusarium oxysporum. There is a pressing need to identify genes that confer host-plant resistance to this extremely destructive disease of watermelon as a means to reduce the damaging losses. Using watermelon populations made up of individuals that differ in response to infection by race 1 of Fusarium and an advanced DNA technology system known as Kompetitive Allele Specific Polymerase Chain Reaction markers, ARS researchers at Charleston, South Carolina, were able to identify gene sequences associated with resistance. These were then used to develop specific markers that identify plants containing a major gene conferring resistance to the race 1 fungus. The resulting Kompetitive allele markers are of great interest to watermelon breeders and geneticists and should be useful to any watermelon breeder working to accelerate the development of elite watermelon cultivars resistant to Fusarium.

Review Publications
Ren, Y., Guo, S., Zhang, J., He, H., Sun, H., Tian, S., Gong, G., Zhang, H., Levi, A., Tadmor, Y., Xu, Y. 2017. A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon. Plant Physiology. 176(1):836-850. https://doi:10.1104/pp.17.01290.
Branham, S., Vexler, L., Meir, A., Tzuri, G., Frieman, Z., Levi, A., Wechter, W.P., Tadmor, Y., Gur, A. 2017. Genetic mapping of a major co-dominant QTL associated with B-carotene accumulation in watermelon. Molecular Breeding. 37:146.
Levi, A., Ling, K. 2017. USVL-380, A zucchini yellow mosaic virus resistant watermelon breeding line. HortScience. 52(10):1448-1450. https://doi:10.21273/HORTSCI12292-17.
Saminathan, T., Garacia, M., Ghimire, B., Lopez, C., Bodunrin, A., Balagurusamy, N., Nimmakayala, P., Huber, D., Levi, A., Reddy, K. 2018. Metagenomic and metatranscriptomic analysis of the microbiome of watermelon fruits. Frontiers in Plant Science. 9:1-4.
Stansell, Z., Bjorkman, T., Branham, S., Couillard, D.M., Farnham, M.W. 2017. Use of a quality trait index to increase the reliability of phenotypic evaluations in broccoli. HortScience. 52(11):1490-1495. https://doi:10.21273/HORTSCI12202-17.
Nimmakayala, P., Saminathan, T., Abburi, V.L., Yadav, L.K., Tomason, Y., Levi, A., Weng, Y., Reddy, K.U. 2017. Comparative Genomics of the Cucurbitaceae. In: Grumet, R., Katzir, N., Garcia-Mas, J, editors. Plant Genetics and Genomics: Crops and Models. Springer International Publishing AG. 20:p. 229-240. https://doi:10.1007/7397_2017_2.
Wu, S., Shamimuzzaman, M., Sun, H., Salse, J., Sui, X., Wilder, A.J., Wu, Z., Levi, A., Xu, Y., Ling, K., Fei, Z. 2017. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ringspot virus resistance locus. Plant Journal. 92:963-975.
Cutulle, M.A., Harrison Jr, H.F., Kousik, C.S., Wadl, P.A., Levi, A. 2017. Bottle gourd genotypes vary in clomazone tolerance. HortScience. 52:1687-1691.
Levi, A., Jarret, R.L., Kousik, C.S., Wechter, W.P., Nimmakayala, P., Reddy, U. 2017. Genetic Resources of Watermelon. In: Grumet R., Garcia-Mas J., and Katzir N., editors. Genetics and Genomics of Cucurbitaceae. Springer International Publishing AG 2016. p. 87-110. https://doi:10.1007/7397_2016_34.
Branham, S., Levi, A., Katawczik, M.L., Fei, Z., Wechter, W.P. 2018. Construction of a genome-anchored, high-density genetic map for melon (Cucumis melo L.) and identification of Fusarium oxysporum f. sp. melonis race 1 resistance QTL. Journal of Theoretical and Applied Genetics. 131:829-837. https://doi:10.1007/s00122-017-3039-5.
Branham, S., Farnham, M.W., Robinson, S.M., Wechter, W.P. 2018. Identification of resistance to bacterial leaf blight in the U.S. Department of Agriculture collard collection. HortScience. 53:838-841. https://doi:10.21273/HORTSCI12347-17.
Branham, S., Levi, A., Farnham, M.W., Wechter, W.P. 2017. A genotype-by-sequencing-single nucleotide polymorphism based linkage map and quantitative trait loci (QTL) associated with resistance to Fusarium oxysporum f. sp. niveum race 2 identified in Citrullus lanatus var. citroides. Theoretical and Applied Genetics. 130: 319-330.