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

2020 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
Under Sub-objective 1B, research continued on mapping major Quantitative Trait Loci (QTL) for resistance to downy mildew (DM) and developing 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. Phenotypic data were collected for resistance to DM on two mapping populations. Seeds of F3 filial generation were produced from 52 lines. Under Sub-objective 1C, six Fusarium wilt-resistant lettuce breeding lines released in 2019 continue to be of interest for commercial field tests in Yuma, Arizona, and Salinas, California. Seed were distributed to about 20 national and international requestors. A search for higher level resistance in primitive accessions and closely related, wild relatives was discussed and initiated with collaborators at University of Arizona, Yuma Center for Excellence in Desert Agriculture, and University of Florida. Under 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. A linkage map of the mapping population was developed using SNP-based markers and phenotypic data for lettuce resistance to sclerotia wilt that were collected from two field trials. Seed of 19 F3 filial generation and BC2 generation were produced in a greenhouse. Under Sub-objective 1G, research continued on development of tools for automatic phenotyping of lettuce deterioration. Phenotypic data of deterioration were collected from over 2,000 samples of fresh-cut lettuce. Phenotyping of 150 accessions with FluorCam has been performed. Sub-objective 1H is to develop romaine breeding lines with reduced incidence of tipburn. Maximized telework is preventing field trials to evaluate resistance to tipburn planned for May 2020. Tests will be rescheduled for fiscal year 2021 to evaluate 244 breeding lines. Thirty F1 populations segregating for tipburn resistance are growing in the greenhouse for generation advance and seed increase. Sub-objective 1I is to identify lettuce germplasm with resistance to Impatiens necrotic spot virus (INSV). A retirement in December 2019 left a critical vacancy and is slowing progress. During Maximized telework, the thrips colony (INSV vector) in the greenhouse was lost and is under redevelopment in preparation for continued germplasm screening. Natural INSV infection in late 2019 field trials provided validation of previous greenhouse INSV results in identifying cultivars and experimental germplasm with INSV resistance. Sub-objective 1J is to identify higher levels of resistance to Verticillium dahliae race 2 and develop resistant iceberg lettuce. In the growth room and greenhouse, plant evaluation trials are underway to determine resistance to Verticillium dahliae in three separate populations. In the greenhouse, lines are being selfed to increase seed for continued evaluation. In support of Objective 2 (spinach), crosses and selections were made for resistances to downy mildew, leafminers, and herbicide, as well as horticultural traits by using a recurrent selection method. Several populations showed high levels of resistance to downy mildew and leafminers. In our trials, 10 spinach breeding populations had 0%, and another five populations had under 10% downy mildew incidences, as compared with the susceptible control (‘Viroflay’) with 98% disease incidence. Our results show that the recurrent selection method was very effective for increasing downy mildew resistance in spinach populations. 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 research project (funded by USDA National Institute of Food and Agriculture (NIFA) Agriculture and Food Research Initiative (AFRI) Food Security Grant Program, USDA NIFA Specialty Crop Research Initiative, and USDA Agricultural Marketing Service Specialty Crop Multi-State Grant Program), a postdoc is continuing the investigation of the roles of oospores, transcriptional changes, and biofungicide in downy mildew development in collaboration with a pathologist. In support of Sub-objective 3A, disease reactions of three sets of melon powdery mildew race differentials were compared: 1) commonly used differential set of 13 lines, 2) 21-differential set in a triple septet structure for purposes of establishing a uniform and objective means of describing powdery mildew races, and 3) 12-differential set proposed by the International Seed Federation for detection of emerging and economically important races and European Union (EU) seed testing requirements. There were two tests, one with the Salinas isolate of race S, and the second test with three isolates (races 2, S and U).

Accomplishments
1. Identification of romaine lettuces with reduced browning discoloration for fresh-cut processing. Fresh-cut lettuce is the primary ingredient of the increasingly popular, packaged, ready-to-eat salads; however, discoloration (browning) represents a major challenge that limits the quality and shelf life of packaged lettuce. The lack of effective browning control has resulted in processors relying on modified atmosphere packaging (MAP) to achieve low oxygen atmospheric conditions and maintain the shelf life. ARS researchers in Salinas, California, and Beltsville, Maryland, identified lettuces with limited browning that will be used in the breeding programs and to identify genes associated with limited browning. Lettuce is one of the most valuable fresh vegetables and is in the top ten most valuable crops in the United States, with an annual farm-gate value of over $2.5 billion.

2. Cold tolerance gene in lettuce identified. Extreme weather is becoming more frequent with climate change. Frost damage often occurs in major winter lettuce production areas of Imperial Valley, California and Yuma, Arizona, leading to significant quality and yield losses. Through genomic, physiological, and gene expression analyses, ARS researchers at Salinas, California, discovered that a mutant allele of the LsCBF7 gene causes reduced freezing tolerance in lettuce. The mutation results in a premature stop codon in the gene and was found in 87% of about 500 cultivated lettuce varieties, including all 114 iceberg cultivars tested, but only 9% of 66 accessions of the wild lettuce progenitor Lactuca serriola. The mutation is associated with late bolting, a desirable trait in lettuce, suggesting that the mutant allele was likely selected for and preserved during the domestication or development of lettuce cultivars. These findings provide insight into the evolution and mechanism of cold response and feasibility for the genetic improvement of cold tolerance in lettuce.

3. QTL for resistance to Cucurbit yellow stunting disorder virus (CYSDV) detected in melon. Sweet potato whitefly and Cucurbit yellow stunting disorder virus (CYSDV) virtually eliminated fall melon production in the desert southwest United States. Moderate resistance to CYSDV was identified in a melon accession from India and its inheritance determined. Selection for resistance in naturally infected field tests is confounded by co-infection with other whitefly transmitted viruses. ARS researchers at Salinas, California, used a CYSDV-specific virus titer assay to identify a Quantitative Trait Loci (QTL) associated with resistance to CYSDV. The presence of a closely linked marker was confirmed in low-virus titer-segregants from a cross of susceptible x resistant lines, as well as in other resistance sources. These results will facilitate development of CYSDV-resistant melon cultivars for the desert southwest United States.

Review Publications
Simko, I. 2019. Genetic variation and relationship among content of vitamins, pigments, and sugars in baby leaf lettuce. Food Science and Nutrition. 7(10):3317–3326. https://doi.org/10.1002/fsn3.1196.
Adhikari, N.D., Simko, I., Mou, B. 2019. Phenomic and physiological analysis of salinity effects on lettuce. Sensors. 19(21):4814. https://doi.org/10.3390/s19214814.
Sandoya, G.V., Maisonneuve, B., Truco, M.J., Bull, C.T., Simko, I., Trent, M., Hayes, R.J., Michelmore, R.W. 2019. Genetic analysis of resistance to bacterial leaf spot in the heirloom lettuce cultivar Reine des Glaces. Molecular Breeding. 39:160. https://doi.org/10.1007/s11032-019-1072-6.
Mou, B. 2019. ‘USDA Red’ spinach. HortScience. 54(11):2070-2072. https://doi.org/10.21273/HORTSCI14308-19.
Simko, I. 2020. Genetic variation in response to N, P, or K deprivation in baby leaf lettuce. Horticulturae. 6(1):15. https://doi.org/10.3390/horticulturae6010015.
Park, S., Shi, A., Mou, B. 2020. Genome-wide identification and expression analysis of the CBF/DREB1 gene family in lettuce. Scientific Reports. 10:5733. https://doi.org/10.1038/s41598-020-62458-1.
Dong, L., Ravelombola, W., Weng, Y., Qin, J., Zhou, W., Bhattarai, G., Zia, B., Yang, W., Shi, L., Mou, B., Shi, A. 2019. Change in chlorophyll content over time well-differentiated salt-tolerant, moderately salt-tolerant, and salt-susceptible cowpea genotypes. HortScience. 54(9):1477-1484. https://doi.org/10.21273/HORTSCI13889-19.
Kandel, S.L., Subbarao, K.V., Shi, A., Mou, B., Klosterman, S.J. 2019. Evaluation of biopesticides for managing downy mildew of spinach in organic production systems in 2017 and 2018. Plant Disease Management Reports. 13:V171.
Zhu, S., Niu, E., Shi, A., Mou, B. 2019. Genetic diversity analysis of olive germplasm (Olea europaea L.) with genotyping-by-sequencing technology. Frontiers in Genetics. 10:755. https://doi.org/10.3389/fgene.2019.00755.
Ravelombola, W., Qin, J., Weng, Y., Mou, B., Shi, A. 2019. A simple and cost-effective approach for salt tolerance evaluation in cowpea (Vigna unguiculate) seedlings. HortScience. 54(8):1280-1287. https://doi.org/10.21273/HORTSCI14065-19.
Macias Gonzalez, M., Truco, M.J., Bertier, L., Jenni, S., Simko, I., Hayes, R.J., Michelmore, R.W. 2019. Genetic architecture of tipburn resistance in lettuce. Theoretical and Applied Genetics. 132(8):2209–2222. https://doi.org/10.1007/s00122-019-03349-6.
Inderbitzin, P., Christopoulou, M., Lavelle, D., Reyes-Chin-Wo, S., Michelmore, R.W., Subbarao, K.V., Simko, I. 2019. The LsVe1L allele provides a molecular marker for resistance to Verticillium dahliae race 1 in lettuce. Biomed Central (BMC) Plant Biology. 19:305. https://doi.org/10.1186/s12870-019-1905-9.
Luo, Y., Bornhorst, E., Teng, Z., Zhou, B., Park, E., Turner, E.R., Simko, I. 2019. Identification of romaine lettuce (Lactuca sativa var. longifolia) varieties with reduced browning discoloration for fresh-cut processing. Postharvest Biology and Technology. 156:110931. https://doi.org/10.1016/j.postharvbio.2019.110931.
Mamo, B.E., Hayes, R.J., Truco, M., Puri, K.D., Michelmore, R.W., Subbarao, K.V., Simko, I. 2019. The genetics of resistance to lettuce drop (Sclerotinia spp.) in lettuce in a recombinant inbred line population from Batavia Reine des Glaces x Eruption. Theoretical and Applied Genetics. 132(8):2439-2460. https://doi.org/10.1007/s00122-019-03365-6.
Simko, I. 2020. Predictive modeling of a leaf conceptual midpoint quasi-color (CMQ) using an artificial neural network. Sensors. 20(14):3938. https://doi.org/10.3390/s20143938.
Sthapit Kandel, J., Peng, H., Hayes, R.J., Mou, B., Simko, I. 2020. Genome-wide association mapping reveals loci for shelf life and developmental rate of lettuce. Theoretical and Applied Genetics. 133:1947–1966. https://doi.org/10.1007/s00122-020-03568-2.