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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

2019 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
For the watermelon portion of this project falling under Objective 1A and 1B, we have collaborated with a private seed company to generate genetic populations of a wild type watermelon segregating for resistance to Fusarium wilt race 2, which is considered the most damaging watermelon disease problem in the USA. We collaborated with a Research Plant Pathologist at Charleston to evaluate this population for relative resistance to race 2, and also for resistance to papaya ring spot virus (another significant disease problem in watermelon). Using advanced DNA technologies we were able to identify a region of one chromosome that contains a gene conferring Fusarium wilt resistance. We also identified a region that confers resistance to papaya ringspot virus. We have been developing DNA markers associated with the resistances that will aid plant breeders working to move the wilt and ringspot resistances from the wild to the cultivated type watermelon. We also conducted a DNA marker study and identified DNA sequences closely linked to a unique resistance to a different race (1) of Fusarium wilt. These latter sequences are located on a specific segment of watermelon chromosome 1, confirming previous results. These DNA sequences are also being converted to markers that are easier to analyze and that will be useful in breeding programs working to incorporate wilt resistance into the genetic background of watermelon cultivars. In separate watermelon studies relative to Objective 1C, we have been developing breeding lines showing resistance to another problem virus, the zucchini yellow mosaic virus Florida strain that is also very damaging to watermelon. In a collaboration with a seed company we screened a genetic population segregating for resistance to zucchini yellow mosaic virus and used genomic technologies to identify the region on a particular chromosome that confers resistance to this virus. We are developing DNA markers that can be useful in helping to readily move 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 papaya ringspot virus, are being used to develop resistant germplasm lines and genetic populations for genetic mapping of loci associated with resistance to this problem virus. We have completed the sequencing of the desert watermelon which is considered the progenitor of the cultivated type sweet watermelon and have identified over 800 gene sequences that have been lost during the evolution and domestication of the sweet desert watermelon. Many of the lost gene sequences that still persist in the desert watermelon are known to be associated with resistance to diseases and other pests. We are currently crossing the desert and cultivated types and developing genetic populations and breeding lines that should be useful for plant breeders to enhance disease resistance in modern watermelon cultivars. Through our recent CucCAP project “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops” we collaborated with scientists at Michigan State, North Carolina State, and Cornell on sequencing the entire genome of the principle American watermelon cultivar “Charleston Gray”. We also sequenced the genome of 1,365 watermelon accessions and successfully used the data to identify gene loci associated with resistance to major diseases of watermelon, including powdery mildew, Papaya ringspot virus, and bacterial fruit blotch. The data from this study are available on the Cucurbit Genome Database (CuGenDB) website, and are being used by seed companies for improving disease resistance in elite watermelon varieties. For the broccoli portion of this project falling under Objective 2, an additional cycle of breeding broccoli for high temperature tolerance 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 conducted in the fall and spring have allowed the project to assess the performance of heat tolerant broccoli lines under conditions more favorable for head development. We continue evaluating results from those trials, and those findings will help determine which lines are most stable and have the greatest commercial potential. A separate genomic study focused on heat tolerance was conducted in which pooled groups of heat tolerant and nontolerant broccoli lines from a segregating population were analyzed using a DNA marker technique called QTL-seq. Results from this study identified new sequences associated with heat tolerance that are different from others identified previously. Bioinformatics analysis of this data has been completed, and results have identified new candidate genes that may confer heat tolerance. These sequences will be confirmed in future marker-assisted breeding studies. In separate broccoli work focused on identifying vigorous inbreds that produce head yield and quality comparable to commercial hybrids, about 15 inbreds were grown in the greenhouse and seed supplies were increased for all of them. The resulting seed will be used to grow plants for upcoming trials in the fall and beyond. In work falling under Objective 3, numerous plant introductions of B. rapa were evaluated for response to inoculation with the bacterium Pseudomonas cannabina pv. alisalensis (Pca) and several resistant accessions were identified. Individual resistant plants for certain accessions were moved to a cooler, allowed to vernalize, and then placed in a greenhouse where they flowered and were self-pollinated. Of particular interest were several accessions that had horticultural traits similar to turnip greens. Separate segregating populations of B. rapa resulting from crosses of a field-resistant line resembling Chinese cabbage and plants from a turnip green cultivar were evaluated in the field, and plants that looked most like turnip greens were selected, moved to a greenhouse, and allowed to self-pollinate. The new lots of seed will be grown out, and seedlings will be tested for response to inoculation with Pca. In a related study, we evaluated the response of about 50-60 different collard lines (S1s) derived by selfing resistant plants observed among many screened from the original accessions obtained from a collection of Brassica plant introductions. New resistant selections were identified among evaluated lines, they were moved to a cooler to be vernalized, and after about 10 weeks moved to a greenhouse where they were allowed to flower. All of these selections were selfed by hand to generate S2 seed which will be tested an additional generation for response to inoculation with Pca. This work could ultimately lead to the identification of a blight resistant collard. Relative to the subordinate project on Development of an East Coast Broccoli Industry, six ARS broccoli hybrids were sent to the Principle Investigator at Cornell for inclusion in the 2019 Quality trials. Additionally, seed of four hybrids were sent for on-farm yield trials in Florida, Georgia, North Carolina, Virginia, New York and Maine. During the winter of 2018-19, select broccoli inbreds were cross-pollinated in the greenhouse to generate adequate seed supplies of specific hybrids for testing in 2020. Seed supplies of select inbreds were also increased significantly during the winter pollinating season. In addition, five outdoor cages were used to generate seed of five specific hybrids. All of the ARS hybrids input into the Quality trials are being evaluated for warm season adaptation by cooperating public scientists in Florida, South Carolina, North Carolina, New York, and Maine. In separate work under a Cooperative Research and Development Agreement, this project 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 a health-promoting compound called glucoraphanin. Seed production was conducted in the Yuma Valley in Arizona and in the Central Valley of California during the winter months. Selections from new segregating broccoli populations were made at Charleston and plants identified in the field were moved to a greenhouse and allowed to self-pollinate independently. The resulting F3 seed will be grown in cages in Yuma during the upcoming winter to identify plants with high seed yield.

1. Elucidating additional 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. The likelihood of this environmental stress occurring in a given location or season is typically 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. Unfortunately, improvement of heat tolerance in broccoli has been limited by a lack of genetic knowledge about the trait. ARS scientists at Charleston, South Carolina, developed a unique broccoli population segregating for heat tolerance, then evaluated the divergent lines (e.g., the most tolerant versus the least tolerant) for their ability to produce good quality heads during two summer seasons. Additionally, these researchers used a new DNA sequencing technique in this work to identify DNA markers associated with the production of quality heads, identifying two new gene sequences associated with the tolerance trait that were not previously identified. These new markers are of great interest to public and private broccoli breeders working to accelerate the development of heat tolerant cultivars.

2. The sequencing and assembly of the principle American watermelon variety ‘Charleston Gray’. There has been limited data available about the genomics of watermelon and about the genetic diversity among the United States watermelon Plant Introductions maintained by the USDA. Thus, ARS scientists working in Charleston, South Carolina, collaborated with scientists at Cornell University to sequence and assemble the genome of the principal American watermelon variety ‘Charleston Gray’. This team worked collectively through the Specialty Crops Research Initiative project entitled “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops” to generate sequencing data for 1,365 watermelon introductions. They successfully used the resulting data in genome-wide association studies to identify gene sequences associated with resistance to several major diseases of the crop, including powdery mildew, Papaya ringspot virus, and bacterial fruit blotch. The data generated by 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 varieties.

Review Publications
Branham, S., Wechter, W.P., Lambel, S., Massey, L.M., Ma, M., Fauve, J., Farnham, M.W., Levi, A. 2018. QTL-seq and marker development for resistance to Fusarium oxysporum f. sp. niveum race 1 in cultivated watermelon. Molecular Breeding. 38:139.
Oren, E., Tzuri, G., Vexler, L., Dafna, A., Meir, A., Faigenboim, A., Kenigswald, M., Portnoy, V., Schaffer, A.A., Levi, A., Buckler IV, E.S., Katzir, N., Burger, J., Tadmor, Y., Gur, A. 2019. The multi-allelic APRR2 Gene is associated with fruit pigment accumulation in melon and watermelon. Journal of Experimental Botany.
Wu, S., Wang, X., Reddy, U., Sun, H., Bao, K., Patel, T., Oritz, C., Abburi, L., Nimmakayala, P., Branham, S., Wechter, W.P., Massey, L.M., Ling, K., Kousik, C.S., Hammar, S.A., Tadmor, Y., Portnoy, V., Gur, A., Katzir, N., Guner, N., Davis, A., Hernandez, A.G., Wright, C.L., McGregor, C., Jarret, R.L., Xu, Y., Zhang, X., Wehner, T.C., Grumet, R., Levi, A., Fei, Z. 2019. Genome of ‘Charleston Gray’, the principal American watermelon cultivar, and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection. Plant Biotechnology Journal.
Kantor, M., Levi, A., Thies, J.A., Guner, N., Kantor, C., Parnham, S., Boroujerdi, A. 2018. NMR analysis reveals a wealth of metabolites in root-knot nematode resistant roots of Citrullus amarus watermelon plants. Journal of Nematology. 50(3)303-316.
Simmons, A.M., Jarret, R.L., Cantrell, C.L., Levi, A. 2019. Citrullus ecirrhosus: Wild source of resistance against Bemisia tabaci (Hemiptera: Aleyrodidae) for cultivated watermelon. Journal of Economic Entomology.
Branham, S., Levi, A., Wechter, W.P. 2019. QTL mapping identifies novel source of resistance to Fusarium wilt race 1 in Citrullus amarus. Plant Disease.
Zheng, Y., Wu, S., Bai, Y., Sun, H., Jiao, C., Guo, S., Zhao, K., Blanca, J., Zhang, Z., Huang, S., Xu, Y., Weng, Y., Mazourek, M., Reddy, U., Ando, K., McCreight, J.D., Schaffer, A.A., Burger, J., Tadmor, Y., Katzir, N., Tang, X., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, W.P., Levi, A., Garcia-Mas, J., Grumet, R., Fei, Z. 2018. Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops. Nucleic Acids Research. 47(D1):1128-1136.
Branham, S., Levi, A., Katawczik, M.L., Wechter, W.P. 2019. QTL mapping of resistance to bacterial fruit blotch in Citrullus amarus. Theoretical and Applied Genetics.
Rutter, W.B., Kousik, C.S., Thies, J.A., Farnham, M.W., Fery, R.L. 2018. PA-593: A root-knot nematode resistant sweet cherry-type pepper. Horticultural Science. 53(12):1922-1923.
Branham, S., Farnham, M.W. 2019. Identification of heat tolerance loci in broccoli through bulked segregant analysis using whole genome resequencing. Euphytica. 215:34.