Research Project: Genetic Improvement of Lettuce, Spinach, Celery, Melon, and Related Species

Location: Crop Improvement and Protection Research

2024 Annual Report

Objectives
Objective 1: Develop genetic resources and germplasm resistance to key pests and pathogens and abiotic stresses of lettuce, spinach, celery, and melon. Sub-objective 1.A: Breed for resistances to fungal, bacterial, and viral diseases and disorders in lettuce. Sub-objective 1.B: Develop durable resistance to downy mildew in spinach. Sub-objective 1.C: Improve celery for resistance to Fusarium oxysporum f.sp. apii (Foa) races 2 and 4. Sub-objective 1.D: Breed melon for resistance against powdery mildew, virus, and whitefly. Objective 2: Develop new procedures and technologies to evaluate and enhance postharvest quality and shelf life of fresh cut leafy greens, especially lettuce. Objective 3: Improve content and bioavailability of phytonutrients or fiber of leafy vegetables for improved impact on human health nutrition and composition of the gut microbiome. Sub-objective 3.A: Characterize polyphenol and fiber content among lettuce cultivars. Sub-objective 3.B: Breed red spinach for improved nutrient content and disease resistance. Objective 4: Characterize the composition and effects of environment on the microbiota found on leafy vegetables.

Approach
Objective 1: Sub-objective 1A: Map major QTLs for resistance to downy mildew using a genome-wide association mapping (GWAS) approach and develop breeding lines with the improved resistance to lettuce drop and downy mildew. Characterize resistance to Fusarium, Verticillium, and Pythium wilts to develop improved lettuce germplasm. Cross corky root-resistant variety ‘Glacier’ and wild species L. serriola to sources of other disease resistance and select for combined resistances to corky root, leafminer, downy mildew, lettuce mosaic virus, tipburn, and horticultural and nutritional traits in different types of lettuce. Characterize resistance to impatiens necrotic spot virus to develop improved lettuce germplasm. Develop controlled environment and molecular assays to characterize tipburn resistance in lettuce. Sub-objective 1B: Breed open-pollinated (OP) spinach with resistance to all prevalent downy mildew races through crosses in isolators, recurrent selection, and replicated field trials. Sub-objective 1C: Develop celery germplasm with resistance to Foa races 2 and 4 through disease assays, selection, and self-pollination. Sub-objective 1D: Breed western U.S. shipper type melon resistant to powdery mildew races 1, 2, 3.5, 5, and S. Identify and characterize resistance in melon to whitefly-transmitted cucurbit chlorotic yellows virus (CCYV). Characterize antixenosis to sweetpotato B biotype whitefly in melon. Objective 2: Fine-map the locus for slow decay of fresh-cut lettuce, map QTLs for limited oxidative browning, and develop lettuce breeding lines with high postharvest quality. Objective 3: Sub-objective 3A: Characterize polyphenol and fiber content among lettuce cultivars to understand genotype x environment interactions and to identify genetically stable high polyphenol cultivars in replicated greenhouse assays. Sub-objective 3B: Improve the betacyanin content, antioxidant capacity, and downy mildew resistance of different types of spinach through crossing, recurrent selection, and field trials. Objective 4: Determine the relationships in the phyllosphere community among leafy vegetables, nearby weeds, and the soil microbiota by isolating genomic materials from the phyllosphere and rhizosphere for sequencing.

Progress Report
This report documents progress for project 2038-21530-003-000D, titled, “Genetic Improvement of Lettuce, Spinach, Celery, Melon, and Related Species”, which started in April 2023. In support of Sub-objective 1.A, Goal 1.A.3, ARS researchers in Salinas, California, conducted greenhouse tests in February and April to evaluate commercial lettuce varieties and internationally recognized Fusarium differential varieties to characterize reports of “new races” in commercial fields. Germplasm was inoculated with multiple Fusarium isolates representing race 1 and the new variant. Discussions with ARS and university researchers is leading to coordinated efforts to biological and molecular characterization of these and other isolates and a clear direction for resistance breeding. The researchers planted a field trial in June to evaluate USDA breeding lines and check varieties under Verticillium race 1 and Fusarium race 1. Selections for disease resistance and horticultural traits will be conducted in September and promising lines will be moved forward for germplasm development and release. Previously selected lines were crossed in the greenhouse and seed generations were advanced for future evaluation. ARS researchers at Salinas, California, in collaboration with university researchers, optimized and validated a Pythium wilt inoculation protocol to evaluate germplasm for resistance. For Sub-objective 1.A, Goal 1.A.4, lettuce breeding lines in advanced generations were tested in field trials with control varieties and commercial cultivars. The corky root and leafminer resistances of the breeding lines were similar to or better than resistant controls, while their plant weight, height, core length, tipburn, and downy mildew resistance were comparable or better than control cultivars. Two green leaf, one red leaf, and two romaine lettuce breeding lines with resistances to leafminer, corky root, downy mildew, and/or tipburn have been publicly released and published. Under Sub-objective 1.A, Goal 1.A.5, ARS researchers in Salinas, California, conducted greenhouse tests in January and May to evaluate selected breeding lines, commercial varieties, wild accessions, and check varieties for resistance to impatiens necrotic spot virus (INSV). ARS researchers at Salinas, California, identified the region of the genome linked to INSV resistance from ‘Eruption’, which will advance breeding and development efforts towards germplasm release. Previously selected lines were crossed in the greenhouse and seed generations were advanced for future evaluation. The researcherswill plant fields trials in June and August to evaluate commercial varieties, USDA breeding lines, and check varieties for resistance to INSV. They will harvest in early FY25. Supporting Sub-objective 1.A Goal 1.A.6, ARS researchers evaluated growth chamber conditions to induce tipburn in lettuce under controlled environments. The researchers tested two tipburn susceptible lines at five temperature conditions and two watering regimes and identified conditions that appear more conducive to this physiological disorder. Controlled environment conditions will be confirmed and optimized to consistently produce tipburn, which will allow for selections of tipburn-resistance in USDA breeding lines in the future. For Sub-objective 1.B, thirty-four spinach populations of western and oriental types were planted in the field to confirm the previous results, and 10-50 plants were selected from each population to produce seeds in isolators. Some plants were selfed. Replicated field trials were conducted in the field to evaluate the downy mildew resistance of 28 populations, and resistant plants were selected for seed production. In support of Sub-objective 1.C, ARS researchers planted approximately 200 seeds from a previously self-pollinated celery germplasm collection and assayed for disease resistance in a field infested with Fusarium oxysporum forma speciales. apii (FOA) race 4 in December. Twenty-one plants were selected for disease resistance and agronomic traits, and vernalized to induce bolting and flowering in January. The ARS researchers will self-pollinate these lines and collect seed in early FY25, which will advance breeding efforts to release germplasm with FOA race 2 and race 4 resistance. Under Sub-objective 1.D, a melon core collection/set for genetic analyses and screening of economically important traits was previously established. It consists of 384 diverse melons (from North America, Europe, Africa, Central and East Asia and India) and represents approximately 99% of the genetic diversity in approximately 2,083 melon accessions mostly contained in the USDA, National Plant Germplasm System (NPGS) melon collection. The set is in the process of being made more uniform via a process of single seed descent for submission to USDA, NPGS. Supporting Objective 2, mating of closely related recombinant inbred lines with genetic differences in the qSL4 region was conducted and F1 seeds were successfully produced. Under Sub-objective 3.A, ARS researchers in Salinas, California, planted 52 lettuce varieties in triplicate in the greenhouse and assayed for total polyphenol content. Samples from a subset of cultivars were sent to a private contracting laboratory to evaluate total dietary fiber content, which is expected to be returned in August. This work will advance the understanding of existing content and bioavailability of phytonutrients and fiber, which is necessary to improve the content of phytonutrients and fiber for improved impact on human health and the microbiome. In support of Sub-objective 3.B, ARS researchers continued to improve the color, betacyanin content, and disease resistance of red spinach by conducting recurrent selection in 44 populations (20-40 plants selected from each population of 400-800 plants) and self-pollinating individual plants. For Objective 4, ARS researchers identified six field sites throughout the Salinas Valley to plant cultivated and wild lettuce transplants in order to describe the effect of the environment on the microbiota of the leaves. The transplants were developed in June and will be moved to the field in mid-July. Methods for isolating genomic material from the phyllosphere and rhizosphere were tested, and a standard protocol will be adopted for DNA isolation of microbial communities on the leaf and within the soil.

Accomplishments
1. Release of ‘NutriHi’ lettuce with high nutrient content. Lettuce generally has lower nutritional value than spinach, which is considered a “super food” due to its high nutrient content. Spinach, on the other hand, is also known to have a high content of oxalic acid that gives an astringent taste and may combine with minerals to form insoluble oxalate crystals that reduce the bioavailability and absorption of calcium and iron in diets and may deposit in the kidneys of susceptible people as a common form of kidney stones, leading to much lower consumption of spinach than lettuce. By using conventional breeding techniques, ARS researchers at Salinas, California, developed and released a leaf lettuce variety ‘NutriHi’ that had 100-107% more vitamin A and vitamin C and 35-158% more minerals than other lettuce cultivars in multiple field trials over five years. In many trials, the vitamin and mineral concentrations of ‘NutriHi’ lettuce were similar to or even higher than spinach. ‘NutriHi’ performed well in the Salinas Valley, the most important lettuce production region in the United States, producing a high percentage of plants of adequate size, shape, and uniformity. Increasing the nutrient density of food is important to human health and well-being, especially for the most consumed fresh-market vegetable in the United States – lettuce.

2. Field trials demonstrate resistance to Pythium wilt and impatiens necrotic spot virus (INSV) in lettuce. Co-infection by Pythium wilt and impatiens necrotic spot virus (INSV) is growing in incidence and severity and significantly impacting lettuce production in California. Genetic resistance through breeding efforts is the most effective control. ARS researchers in Salinas, California, with collaborators from University of California Cooperative Extension and California State University Monterey Bay, coordinated field trials in Pythium infested soil under natural INSV pressure to demonstrate new cultivars from private breeding companies with improved resistance to Pythium wilt and INSV. Stakeholders use this information to make selections on which lettuce varieties to purchase and grow commercially.

3. Field trials demonstrate resistance to Fusarium oxysporum forma speciales apii (FOA) race 2 in Santa Maria, California, and to race 4 in Camarillo, California. FOA race 2 and race 4 are soil-borne pathogens that cause Fusarium wilt disease of celery in California, which significantly impacts yield from infested fields. FOA race 4 is highly virulent and can destroy a crop. There are no effective fungicides available, and therefore genetic resistance through breeding is the most effective methods of control. ARS researchers in Salinas, California, in collaboration with researchers from University of California Agriculture and Natural Resources from three counties coordinated field trials in two locations to evaluate new cultivars from public and private breeding companies with improved resistance to FOA race 2 and race 4. Growers, shippers, and other stakeholders attended field days to view the trials and use this information to evaluate new and unreleased germplasm.

Review Publications
Simko, I., Subbarao, K.V., Hayes, R.J. 2023. Breeding lettuce for resistance against Sclerotinia minor. HortScience. 58(12):1526–1532. https://doi.org/10.21273/HORTSCI17399-23.
Cho, E., Gurdon, C., Zhao, R.B., Peng, H., Pouley, A., Raskin, I., Simko, I. 2023. Phytochemical and agronomic characterization of high-flavonoid lettuce lines grown under field conditions. Plants. 12(19). Article 3467. https://doi.org/10.3390/plants12193467.
Simko, I., Zhao, R.B. 2023. Phenotypic characterization, plant growth and development, genome methylation, and mineral elements composition of neotetraploid lettuce (Lactuca sativa L.). Frontiers in Plant Science. 14. Article 1296660. https://doi.org/10.3389/fpls.2023.1296660.
Ewing, E.E., Weeden, N.F., Simko, I. 2024. Proanthocyanidins: Key for resistance to Globisporangium (formerly Pythium) seed rot of pea. Journal of the American Society for Horticultural Science. 149(1):37-49. https://doi.org/10.21273/JASHS05340-23.
Richardson, K.L., Nayak, S., Hasegawa, D.K., Eriksen, R.L. 2024. Evaluation of lettuce germplasm for resistance to impatiens necrotic spot virus. Euphytica. 220. Article 33. https://doi.org/10.1007/s10681-023-03285-z.
Ravelombola, W., Dong, L., Barickman, T.C., Xiong, H., Manley, A., Cason, J., Pham, H., Zia, B., Mou, B., Shi, A. 2023. Genetic architecture of salt tolerance in cowpea (Vigna unguiculata (L.) Walp.) at seedling stage using a whole genome resequencing approach. International Journal of Molecular Sciences. 24(20). Article 15281. https://doi.org/10.3390/ijms242015281.
Simko, I. 2023. Differentially methylated genomic regions of lettuce seeds relate to divergence across morphologically distinct horticultural types. AoB Plants. 15(5). Article plad060. https://doi.org/10.1093/aobpla/plad060.
Simko, I., Sthapit Kandel, J., Peng, H., Zhao, R.B., Subbarao, K.V. 2023. Genetic determinants of lettuce resistance to drop caused by Sclerotinia minor identified through genome-wide association mapping frequently co-locate with loci regulating anthocyanin content. Theoretical and Applied Genetics. 136. Article 180. https://doi.org/10.1007/s00122-023-04421-y.
Nayak, S., Richardson, K.L. 2023. Inheritance of partial resistance to isolate VdLs17 of Verticillium dahliae within Lactuca spp. Plant Disease. 107(12):3868-3876. https://doi.org/10.1094/PDIS-09-22-2194-RE.
Peng, H., Luo, Y., Teng, Z., Zhou, B., Pearlstein, D.J., Wang, D., Turner, E.R., Nou, X., Wang, T.T., Tao, Y., Fonseca, J.M., Simko, I. 2024. Genome-wide association mapping reveals loci for enzymatic discoloration on cut lettuce. Postharvest Biology and Technology. 207. Article 112577. https://doi.org/10.1016/j.postharvbio.2023.112577.
Klosterman, S.J., Clark, K.J., Anchieta, A.G., Kandel, S.L., Mou, B., McGrath, M.T., Correll, J.C., Shishkoff, N. 2023. Transmission of spinach downy mildew via seed and infested leaf debris. Plant Disease. 108(4):951-959. https://doi.org/10.1094/PDIS-06-23-1225-RE.
Simko, I. 2023. Dataset on the single nucleotide variation in diversity panel of 500 lettuce accessions genotyped with tunable genotyping-by-sequencing (tGBS) method. Data in Brief. 49. Article 109419. https://doi.org/10.1016/j.dib.2023.109419.
Chen, Y., Xiong, H., Ravelombola, W., Bhattarai, G., Barickman, C., Alatawi, I., Phiri, T.M., Chiwina, K., Mou, B., Tallury, S., Shi, A. 2023. A genome-wide association study reveals region associated with seed protein content in cowpea. Plants. 12(14). Article 2705. https://doi.org/10.3390/plants12142705.
Sandoya, G., Lafta, A., Mou, B. 2024. Heat-tolerant lettuce germplasm (Lactuca sativa L.) identified in romaine and butterhead types for warmer plantings. HortScience. 59(2):151-163. https://doi.org/10.21273/HORTSCI17368-23.
Park, S., Shi, A., Meinhardt, L.W., Mou, B. 2024. Genome-wide characterization and evolutionary analysis of the AP2/ERF gene family in lettuce (Lactuca sativa). Scientific Reports. 13:21990. https://doi.org/10.1038/s41598-023-49245-4.
George, A.S., Simko, I., Brandl, M. 2024. Identification and characterization of lettuce cultivars with high inhibitory activity against the human pathogen Escherichia coli O157:H7: Toward a plant-intrinsic hurdle approach to microbial safety. Postharvest Biology and Technology. 211. Article 112816. https://doi.org/10.1016/j.postharvbio.2024.112816.
Park, S., Shi, A., Mou, B. 2024. Low frequency of the wild-type freezing-tolerance LsCBF7 allele among lettuce population suggest negative selection during domestication and breeding. Theoretical and Applied Genetics. 137:135. https://doi.org/10.1007/s00122-024-04643-8.
Xiong, H., Chen, Y., Ravelombola, W., Mou, B., Sun, X., Zhang, Q., Xiao, Y., Tian, Y., Luo, Q., Alatawi, I., Chiwina, K.E., Alkabkabi, H.M., Shi, A. 2024. Genetic dissection of diverse seed coat patterns in cowpea through a comprehensive GWAS approach. Plants. 13(9). Article 1275. https://doi.org/10.3390/plants13091275.
Lebeda, A., Kristkova, E., Mieslerova, B., Dhillon, N.P.S., McCreight, J.D. 2024. Status, gaps and perspectives of powdery mildew resistance research and breeding in cucurbits. Critical Reviews in Plant Sciences. 43(4):211-290. https://doi.org/10.1080/07352689.2024.2315710.
Eshkabilov, S., Simko, I. 2024. Assessing contents of sugars, vitamins, and nutrients in baby leaf lettuce from hyperspectral data with machine learning models. Agriculture. 14(6). Article 834. https://doi.org/10.3390/agriculture14060834.
Sthapit Kandel, J., Simko, I., Hayes, R.J., Mou, B. 2024. Concentration and retention of ascorbic acid, carotenoids, and sugars in fresh-cut lettuce in modified atmospheric packaging. Horticultural Plant Journal. 11(1):303-313. https://doi.org/10.1016/j.hpj.2023.07.008.