Research Project: Genomics and Genetic Improvement of Disease Resistance and Horticultural Characteristics of Watermelon, Broccoli, and Leafy Brassicas

Location: Vegetable Research

2020 Annual Report

Objectives
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.

Approach
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 researchers at Cornell University in developing the ‘Charleston Gray’ genome sequence which now serves as a reference for all of our genomic/genetic analysis studies. We collaborated with a private seed company to generate large genetic populations segregating for resistance to Fusarium wilt (FW; races 1 and 2), which is considered the most destructive disease of watermelon in the USA and throughout the world. Cooperating with two Plant Pathologists at the Charleston location, the developed populations were analyzed by advanced DNA technologies and used to evaluate for resistance to Fusarium wilt race 2 and for resistance to papaya ring spot virus (another significant disease problem in watermelon). These studies resulted in the identification of one chromosome that contains a gene conferring Fusarium wilt resistance. We also identified a region that confers resistance to papaya ringspot virus. DNA markers associated with the resistance gene loci have been developed and they will prove useful to plant breeders working to incorporate the resistance gene loci from the wild into elite watermelon cultivars. In separate watermelon studies relative to Objective 1C, we have been developing breeding lines resistant to zucchini yellow mosaic virus-Florida strain that causes serious damage to the watermelon crop. We collaborated with a seed company and screened a genetic population segregating for resistance to zucchini yellow mosaic virus (ZYMV). Genomic technologies were then employed to identify a genomic region that contains the eukaryotic elongation factor (eIF4E) gene locus, which was previously determined to be tightly linked to ZYMV-resistance. Specific DNA markers were in turn developed using sequence information and these will be useful in helping to facilitate ready movement of 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 to use in genetic mapping of loci associated with resistance to ZYMV. We have completed sequencing of the desert watermelon which is considered the progenitor of the cultivated-type of sweet watermelon and we have identified over 800 gene sequences that have been lost during the evolution and domestication of the sweet dessert watermelon which is the type of watermelon eaten by consumers. Many of the lost gene sequences that persist in the desert watermelon are known to be associated with resistance to diseases and other pests. We are currently crossing the desert and sweet 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 ongoing CucCAP project “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops” we have collaborated with researchers 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 wild 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 http://cucurbitgenomics.org/, and are being used by seed companies for improving disease resistance in elite watermelon cultivars. 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. 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. Two specific inbred lines (designated USVL156 and USVL160) that are adapted to high temperatures have been prepared for a public release by the end of the current performance period. In separate broccoli work focused on identifying vigorous inbreds that produce head yield and quality comparable to commercial hybrids, about 20 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 have been 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 interest are several accessions that have 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 (S2s) 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 also allowed to flower. All of these selections were selfed by hand to generate S3 generation 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, five ARS broccoli hybrids were sent to the Principle Investigator at Cornell for inclusion in the 2020 Quality trials. Additionally, seed of six hybrids were sent for on-farm yield trials in Florida, North Carolina, Virginia, and New York. During the winter of 2019-20, select broccoli inbreds were cross-pollinated in the greenhouse to generate adequate seed supplies of specific hybrids for testing in 2021. Seed supplies of select inbreds were also increased significantly during the winter pollinating season. In addition, six outdoor cages were used to generate seed of six specific hybrids. All of the ARS hybrids input into the Quality trials are being evaluated for warm season adaptation by cooperating public researchers 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 productions were conducted in the Yuma Valley in Arizona and in the Central Valley of California during the winter months. Selections from segregating F3 broccoli families were made at Charleston and plants identified in the field were moved to a greenhouse and allowed to self-pollinate independently. The same F3 families were also grown in cages in Yuma during the past winter and plants with high seed yield in that environment were selected, kept in the cages, and allowed to self-pollinate in the absence of insect pollinators. The resulting F4 lines will be grown again in similar cages next winter in Yuma.

Accomplishments
1. Identification of gene loci associated with resistance to Fusarium wilt and Papaya ringspot virus in watermelon and the development of DNA markers useful for marker assisted selection. Fusarium wilt race 2 and papaya ring spot virus cause serious damage to the watermelon crop, and there is a need to develop cultivars resistant to these two major diseases. ARS researchers in Charleston, South Carolina, worked in cooperation with other scientists at Cornell University, developed genetic populations segregating for disease resistance, and evaluated the populations for response to inoculation by the causal agents of the two diseases. Using genomic data developed through the efforts of the Specialty Crops Research Initiative project entitled “Leveraging Applied Genomics to Increase Disease Resistance in Cucurbit Crops”, the team successfully identified two gene loci and their respective DNA sequences. One Loci confers resistance to Fusarium wilt race 2 and the other confers resistance to papaya ring spot virus. The data generated by this study are being used by seed companies to improve disease resistance in elite watermelon cultivars.

Review Publications
Guo, S., Zhao, S., Sun, H., Wang, X., Wu, S., Lin, T., Ren, Y., Deng, Y., Zhang, J., Lu, X., Zhang, H., Shang, J., Gong, G., Wen, C., He, N., Li, M., Liu, J., Wang, Y., Zhu, Y., Tian, S., Jarret, R.L., Levi, A., Huang, S., Fei, Z., Liu, W., Xu, Y. 2019. Resequencing of 414 cultivated and wild watermelon accessions identifies selection for fruit quality traits. Nature Genetics. 51:1616–1623. https://doi.org/10.1038/s41588-019-0518-4.
Vijay, J., Suhas, S., Venkata Lakshmi, A., Lopez, C., Padma, N., Levi, A., Umesh, R. 2019. Haplotype networking of GWAS hits for citrulline content indicates positive selection leading to the domestication of watermelon. International Journal of Molecular Sciences [MDPI]. https://doi.org/10.3390/ijms20215392.
Garcia-Lozano, M., Dutta, S., Natarajan, P., Tomason, Y.R., Lopez, C., Katam, R., Levi, A., Nimmakayala, P., Reddy, U. 2019. Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids. Plant Molecular Biology. https://doi.org/10.1007/s11103-019-00942-7.
Branham, S., Wechter, W.P., Ling, K., Chanda, B., Massey, L.M., Zhao, G., Guner, N., Bello, M., Kabelka, E., Fei, Z., Levi, A. 2019. QTL mapping of resistance to Fusarium oxysporum f. sp. niveum race 2 and Papaya ringspot virus in Citrullus amarus. Theoretical and Applied Genetics. 133:677-687. https://doi.org/10.1007/s00122-019-03500-3.
Chiu, Y., Shen, C., Farnham, M.W., Ku, K. 2020. Three-dimensional epicuticular wax on plant surface reduces attachment and survival rate of salmonella during storage. Postharvest Biology and Technology. 166:111197. https://doi.org/10.1016/j.postharvbio.2020.111197.
Cutulle, M., Campbell, H., Couillard, D.M., Ward, B., Farnham, M.W. 2019. Pre transplant herbicide application and cultivation to manage weeds in southeastern broccoli production. Crop Protection. 124:104862. https://doi.org/10.1016/j.cropro.2019.104862.
Farnham, M.W. 2020. Hi-Test: A self-compatible green sprouting broccoli cultivar that yields seed with high levels of glucoraphanin. HortScience. 55(5):743-745. https://doi.org/10.21273/HORTSCI14569-19.
Stansell, Z., Bjorkman, T., Farnham, M.W. 2019. Complex horticultural quality traits in broccoli are illuminated by evaluation of the immortal BolTBDH mapping population. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.01104.