Research Project: Genetics and Breeding of Lettuce, Spinach, Melon, and Related Species to Improve Production and Consumer-related Traits

Location: Crop Improvement and Protection Research

2019 Annual Report

Objectives
The focus of this research program is on quality traits, resistances to diseases, insects and abiotic stresses of lettuce, spinach and melon considered by the respective industries and the scientific community to be the most critical to production. We will develop elite germplasm and cultivars with improved quality and productivity, and new knowledge of the genetics and breeding of lettuce, spinach, and melon. Specifically, during the next five years we will focus on the following objectives. Objective 1: Discover and understand novel sources of resistance in lettuce to priority diseases and insects, tolerance to unfavorable abiotic factors (including physiological defects), and improved phytonutrient content; discover trait-linked molecular markers, and use these resources to develop and release improved lettuce germplasm and/or finished varieties. • Subobjective 1A: Corky Root • Subobjective 1B: Downy Mildew • Subobjective 1C: Fusarium Wilt • Subobjective 1D: Leafminer • Subobjective 1E: Lettuce Drop • Subobjective 1F: Phytonutrients • Subobjective 1G: Postharvest Quality • Subobjective 1H: Tipburn • Subobjective 1I: Impatiens necrotic spot virus • Subobjective 1J: Verticillium Wilt Objective 2: Discover and understand novel sources of resistance in spinach to new and emerging diseases (especially downy mildew) and insects (including leaf miner), and develop and release improved spinach germplasm and/or finished varieties. • Subobjective 2A: Spinach Downy Mildew • Subobjective 2B: Leafminer • Subobjective 2C: Linuron Herbicide Tolerance Objective 3: Discover and understand novel sources of resistance in melon to priority diseases and insect pests, and develop and release improved cantaloupe and honeydew germplasm and/or finished varieties with durable resistance. • Subobjective 3A: Resistance to Powdery Mildew • Subobjective 3B1: Resistance to Sweetpotato Whitefly • Subobjective 3B2: Determine inheritance of antixenosis • Subobjective 3B3: Introgression of Antixenosis

Approach
1A: Corky Root. Approach: Combine resistances to corky root, leafminer, downy mildew, lettuce mosaic virus, & tipburn, & nutritional traits; pedigree selection & backcross for type. 1B: Downy Mildew. Approach: Map QTL in 2 F6 RIL populations & develop breeding lines with improved level of resistance. Cross resistant RIL & accessions; pedigree selection & backcross for type. 1C: Fusarium Wilt. Approach: Develop Fusarium wilt-resistance for the Salinas Valley by crossing advanced resistant desert selections with ‘Salinas’; backcross resistant F2 selection to ‘Salinas’, repeat to BC4F4. 1D: Leafminer Approach: Introgress leafminer resistance to different lettuce types by intercrossing resistance sources, then crossing them with breeding lines for combined resistances. Pedigree selection to F6. 1E: Lettuce Drop. Approach: Map QTL for resistance in a F6 RIL population; develop romaine lettuce with improved resistance using most resistant RIL & other accessions. Pedigree selection & backcross for type. 1F: Phytonutrients. Approach: Improve phytonutrient content of lettuce by crossing high carotenoid, anthocyanin, and antioxidant content sources with elite cultivars. Pedigree selection & backcross for type. 1G: Postharvest Quality. Approach: Develop tools to improve lettuce shelf life by combining automatic phenotyping, mapping & molecular markers for MAS; release breeding lines with extended shelf life. 1H: Tipburn. Approach: Develop romaine breeding lines with reduced incidence of tipburn using pedigree selection and backcrossing of advanced lines; select in desert and coastal environments. 1I: Impatiens necrotic spot virus. Approach: Identify resistance sources in Salinas & Pullman accessions in greenhouse tests; mechanical and thrips inoculations. Cross most resistant with elite cultivars. 1J: Verticillium Wilt. Approach: Identify higher levels of resistance to V. dahliae race 2 in Salinas & Pullman lettuce collection. Cross most resistant accessions with elite cultivars. 2A: Spinach Downy Mildew. Approach: Open-pollinated (OP) seed from resistant hybrid spinach cultivars will be OP with susceptible ‘Viroflay’; recurrent selection to combine resistances in OP lines. 2B: Leafminer. Approach: Breed for leafminer resistance against both stings and mines using recurrent selection starting with highest sources of resistance. 2C: Linuron Herbicide Tolerance. Approach: Recurrent selection to increase tolerance to Linuron in field tests. 3A: Resistance to Powdery Mildew. Approach: Introgress resistance in PI 313970 to races 1, 2, 3.5, 5, and S using F2 and F2:3 selections in greenhouse & field tests. Pedigree selection & backcross for type. 3B1: Resistance to Sweetpotato Whitefly. Approach: Compare antixenosis in 4 accessions using individual & group responses, odor-based assays, electrical penetration graphs, & candidate compounds. 3B2: Determine inheritance of antixenosis. Approach: Determine whether antixenosis in PI 122847 is simply inherited or quantitative using Y-tube assays of F2. 3B3: Introgression of Antixenosis. Approach: Introgress antixenosis in PI 122847 to elite western shipping type melon using backcrossing and inbreeding.

Progress Report
In support of Sub-objectives 1A, 1D, and 1F, researchers continued to make crosses, selections, and seed increases to breed for resistances to corky root, leafminers, and phytonutrient improvement, respectively, and horticultural traits. Breeding lines in advanced generations are being 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 resistances were comparable or better than control cultivars. Several extramural grants were obtained to study the abiotic stress tolerance, resistance to bacterial leafspot, and baby-leaf traits in lettuce. Genotypes with tolerance to heat, drought, and salinity as well as good shelf-life were identified. Physiological and genomic studies were conducted to find the mechanism, genetic control, and molecular markers of stress tolerance in lettuce. A new project was started to investigate the cold tolerance in lettuce. Related to Sub-objective 1B, research continued on mapping major Quantitative Trait Loci (QTL) for resistance to downy mildew and development of lettuce breeding lines with the improved resistance to downy mildew. Linkage maps of two mapping populations were developed and genotyped with Single Nucleotide Polymorphism (SNP) markers. Seed of F2 filial generation were produced from 52 lines. In support of Sub-objective 1E, research was performed to identify and to map major QTL for resistance to lettuce drop and to develop breeding lines of romaine lettuce with the improved resistance to lettuce drop. Linkage map of the mapping population was developed using SNP-based markers. Phenotypic data for lettuce resistance to sclerotia wilt were collected from two field trials. Seed of 23 F2 filial generation and BC1 generation were produced in a greenhouse. In Sub-objective 1G, research continued on development of tools for automatic phenotyping of lettuce deterioration. Phenotypic data of deterioration were collected from over 5,000 samples of fresh-cut lettuce. Linkage map based on SNP markers was constructed and aligned to the physical map of lettuce. A major QTL associated with lettuce deterioration was positioned on the linkage map. Sub-objective 1H is to develop romaine breeding lines with reduced incidence of tipburn. In May 2019, 280 breeding lines were planted in replicated Salinas field experiments to evaluate resistance to tipburn. Ten F3 populations segregating for tipburn resistance were evaluated in replicated field plots in 2019. Selected plants from 2018 field experiments were backcrossed to romaine lines to improve horticultural traits. Sub-objective 1I is to identify lettuce germplasm with resistance to Impatiens necrotic spot virus (INSV). In the fall, 39 F1 progeny from the cross of a resistant and susceptible line and check entries were evaluated in greenhouse experiments for resistance to INSV. During the government shutdown, the thrips colony (INSV vector) was lost and is under redevelopment in preparation for continued germplasm screening. Sub-objective 1J is to identify higher levels of resistance to Verticillium dahliae race 2 and develop resistant iceberg lettuce. Protocols are being refined for inoculation of greenhouse and growth chamber tests to evaluate lines for resistance to Verticillium wilt. Seed is being increased in the greenhouse for further evaluation. Under Sub-objectives 2A, 2B, and 2C, we continued to make crosses and selections to breed for resistances to downy mildew, leafminers, and herbicide, respectively, as well as horticultural traits by using a recurrent selection method. Several populations showed high levels of resistance to downy mildew and leafminers. Selected plants were transplanted into isolators to produce seeds for further rounds of selection. Seeds from isolators are being harvested and cleaned. Researchers also continued grant-funded spinach genomic studies with collaborators at University of Arkansas to find molecular markers for disease resistance and horticultural traits. In another grant-funded research project, a postdoc is continuing the investigation of the roles of oospores and biofungicides in downy mildew development in collaboration with a pathologist.

Accomplishments
1. World's first spinach developed with red leaves. Spinach has always been known as a green leafy vegetable. Although there are currently some “red” spinach cultivars on the market, the red color is limited to the veins of the leaves. ARS researchers at Salinas, California, developed ‘USDA Red’, the world’s first spinach variety with red color on the surface of the leaves. ‘USDA Red’ had 65% higher betacyanin content and 53% higher antioxidant capacity than red-veined spinach cultivars on average in field trials conducted in 2015-2018. The betacyanin adds another punch to a plant already loaded with phytonutrients, making spinach a true “super food”. The red spinach may bring some excitement to the spinach market and attract consumers to the colorful new product, helping increase the consumption of spinach and aid in the fight against obesity.

2. Fusarium wilt-resistant lettuce breeding lines. Fusarium wilt of iceberg lettuce has become a yield limiting factor in three western U.S. lettuce production areas. It is especially serious in the early-fall planting period (mid- to late September) in the Yuma, Arizona, area, and is becoming more important in the Salinas Valley of California. ARS researchers at Salinas, California, in collaboration with University of Arizona and industry, identified a high-level source of resistance to the disease in a Romaine lettuce type that was then used as a source of resistance in a cross with an ARS-developed iceberg lettuce adapted to the Yuma production area. Six related breeding lines developed from the cross material has exhibited resistance to the disease in commercial lettuce fields in Yuma and Salinas. The released germplasm is highly resistant to Fusarium wilt and is variable for plant type that is similar to iceberg; a few lines exhibit leaves with red coloration (anthocyanin).

Review Publications
Ravelombola, W., Shi, A., Qin, J., Weng, Y., Bhattarai, G., Zia, B., Zhou, W., Mou, B. 2018. Investigation on various above-ground traits to identify drought tolerance in cowpea seedlings. HortScience. 53(12):1757-1765. https://doi.org/10.21273/HORTSCI13278-18.
Dong, L., Ravelombola, W., Weng, Y., Qin, J., Bhattarai, G., Zia, B., Zhou, W., Wang, Y., Mou, B., Shi, A. 2019. Seedling salt tolerance for above ground-related traits in cowpea (Vigna unguiculata (L.) Walp). Euphytica. 215:53. https://doi.org/10.1007/s10681-019-2379-4.
Kandel, S.L., Mou, B., Shishkoff, N., Shi, A., Subbarao, K.V., Klosterman, S.J. 2019. Spinach downy mildew: Advances in our understanding of the disease cycle and prospects for disease management. Plant Disease. 103:791-803. https://doi.org/10.1094/PDIS-10-18-1720-FE.
Fletcher, K., Klosterman, S.J., Derevnina, L., Martin, F.N., Bertier, L.D., Koike, S.T., Chin-Wo-Reyes, S., Mou, B., Michelmore, R. 2018. Comparative genomics of downy mildews reveals potential adaptations to biotrophy. BMC Genomics. 19:851. https://doi.org/10.1186/s12864-018-5214-8.
Weng, Y.J., Revelombola, W.S., Yang, W., Qin, J., Zhou, W., Wang, Y.-J., Mou, B., Shi, A.N. 2018. Screening of seed soluble sugar content in cowpea (Vigna unguiculata (L.) Walp). American Journal of Plant Sciences. 9:1455-1466. https://doi.org/10.4236/ajps.2018.97106.
Hayes, R.J., Sandoya, G., Mou, B., Simko, I., Subbarao, K.V. 2018. Release of three iceberg lettuce populations that combined resistance to two soil borne diseases. HortScience. 53(2):247-250. https://doi.org/10.21273/HORTSCI12559-17.
Xiong, H., Qin, J., Shi, A., Mou, B., Wu, D., Sun, J., Shu, X., Wang, Z., Lu, W., Ma, J., Weng, Y., Yang, W. 2018. Genetic differentiation and diversity upon genotype and phenotype in cowpea (Vigna unguiculata L. Walp.). Euphytica. 214:4. https://doi.org/10.1007/s10681-017-2088-9.
Ravelombola, S., Shi, A., Weng, Y., Mou, B., Motes, D., Clark, J., Chen, P., Srivastava, V., Qin, J., Dong, L., Yang, W., Bhattarai, G., Sugihara, Y. 2018. Association analysis of salt tolerance in cowpea (Vigna unguiculata (L.) Walp) at germination and seedling stages. Theoretical and Applied Genetics. 131(1):79-91. https://doi.org/10.1007/s00122-017-2987-0.
Simko, I., Hayes, R.J. 2018. Accuracy, reliability, and timing of visual evaluations of decay in fresh-cut lettuce. PLoS One. 13(4):e0194635. https://doi.org/10.1371/journal.pone.0194635.
Sytar, O., Zivcak, M., Bruckova, K., Brestic, M., Olsovska, K., Hemmerich, I., Rauh, C., Simko, I. 2018. Shift in accumulation of flavonoids and phenolic acids in lettuce attributable to changes in ultraviolet radiation and temperature. Scientia Horticulturae. 239:193–204. https://doi.org/10.1016/j.scienta.2018.05.020.
Simko, I., Hayes, R.J., Truco, M., Michelmore, R.W., Antonise, R., Massoudi, M. 2018. Molecular markers reliably predict post-harvest decay of fresh-cut lettuce in modified atmosphere packaging. Horticulture Research. 5:21. https://doi.org/10.1038/s41438-018-0022-5.
Wintermantel, W.M., Hladky, L.L., Fashing, P.L., Ando, K., McCreight, J.D. 2019. First report of Cucurbit chlorotic yellows virus infecting melon in the New World. Plant Disease. 103(4):778. https://doi.org/10.1094/PDIS-08-18-1390-PDN.
Dhillon, N.P.S., Sanguansil, S., Srimat, S., Laenoi, S., Schafleitner, R., Pitrat, M., McCreight, J.D. 2019. Inheritance of resistance to cucurbit powdery mildew in bitter gourd. HortScience. 54(6):1013–1016. https://doi.org/10.21273/HORTSCI13906-19.
Trierweiler, B., Frechen, M.A., Soukup, S.T., Egert, B., Baldermann, S., Sanguansil, S., Mccreight, J.D., Kulling, S.E., Dhillon, N.P. 2019. Bitter gourd, Momordica charantia L., breeding lines differ in secondary metabolite content according to market type. Journal of Applied Botany and Food Quality. 92:106-115. https://doi.org/10.5073/JABFQ.2019.092.015.
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. https://doi.org/10.1093/nar/gky944.