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

2023 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
This is the final report for project 2038-21530-002-000D, Genetics and Breeding of Lettuce, Spinach, Melon, and Related Species to Improve Production and Consumer-related Traits, which has been replaced by new project 2038-21530-003-000D, Genetic Improvement of Lettuce, Spinach, Celery, Melon, and Related Species. For additional information, see the new project report. In support of Sub-objectives 1A, 1D and 1F, ARS researchers in Salinas, California, made crosses, selections, and seed increases to breed for resistances to leafminers, corky root, nutritional improvement, appearance, and horticultural traits. Backcrosses were made when necessary to improve horticultural or resistance traits. 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. A breeding line had significantly higher vitamin A, vitamin C, and mineral concentrations than commercial cultivars tested, and is ready to be released. 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. In support of Sub-objective 1B, research was performed on mapping major Quantitative Trait Loci (QTL) for resistance to downy mildew (DM) 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. Seeds of F3 filial generation were produced from 52 lines. Seeds of F4 filial generation were produced from 27 lines. Phenotypic data were collected for resistance to DM on two mapping populations (years 1-3). Results were published in two peer-reviewed papers. In support of Sub-objective 1C, high level resistance to Fusarium wilt was transferred from the Romaine cultivar King Louie to semi-iceberg lettuce and released in the form of six breeding lines (PI 691904-691909) for additional breeding by commercial breeders and for combining with Verticillium resistance. 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 per year in years 1 and 2, and one trial per year in years 3-4. Seed of 23 F2 filial generation and BC1 generation were produced in a greenhouse in year 1, 19 F3 filial generation and BC2 generation in year 2, and 9 F4 filial generation were produced in year 3. A release notice was written, and seeds were deposited to the U.S. National Plant Germplasm System. Results were published in one peer-reviewed paper. In support of Sub-objective 1G, research was performed to develop 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. Phenotypic data of deterioration were collected from another set of 2,000 samples of fresh-cut lettuce and phenotyping of 150 accessions with FluorCam was performed. Phenotypic data of deterioration were subsequently collected from over 850 additional samples of fresh-cut lettuce and phenotyping of 500 accessions with FluorCam was performed. Finally, phenotypic data of deterioration were collected from another set of 1,000 samples of fresh-cut lettuce. Results were published in two peer-reviewed papers. Sub-objective 1H is to develop romaine breeding lines with reduced incidence of tipburn. Over five years we evaluated 915 USDA breeding lines in Salinas field experiments to evaluate and select for resistance to tipburn and desirable horticultural traits. 114 lines were selected and advanced in the breeding program. 30 lines were backcrossed to romaine lines to improve horticultural traits in preparation for germplasm release. Beginning in 2020, incidence of impatiens necrotic spot virus (INSV) in the field made evaluating for tipburn resistance increasingly difficult. The virus causes significant changes to plant growth and morphology and masks symptoms of tipburn. In the next project plan, we will develop tools to select for tipburn resistance in controlled greenhouse and growth chamber conditions to continue meeting our objective to develop romaine breeding lines with reduced incidence of tipburn. Sub-objective 1I is to identify lettuce germplasm with resistance to INSV, a thrips-vectored orthotospovirus that causes significant economic damage on lettuce in coastal California. In the last five years we developed a viruliferous thrips population for greenhouse germplasm evaluation for INSV resistance. We built on existing protocols to develop a high-throughput method for evaluating large numbers of individuals for INSV resistance in the greenhouse. We planted early- and late-season field trials to capture variation in disease pressure throughout the season. We used these tools to evaluate large numbers of commercial varieties, USDA breeding lines, and wild accessions. We identified a few resistant lines and started crossing with elite romaine and crisphead types. We evaluated a segregating mapping population in the field and greenhouse to identify linked molecular markers. In the next project plan, we will continue this work to release improved germplasm with resistance to INSV and develop marker assisted selection genomic tools. Sub-objective 1J is to identify higher levels of resistance to Verticillium dahliae race 2 and develop resistant iceberg lettuce. Over five years, we evaluated thousands of individuals of 11-G99-1-1 x PI 171674 for resistance to V. dahliae Race 2 in the greenhouse and growth room. The 11-G999-1-1 x PI 171674 population combines two Race 2 resistant sources to identify progeny more resistant than either parent. We recorded verticillium vascular and foliar symptoms, bolting date, date of first flower, chlorophyll content, leaf color, and leaf margin type. We identified transgressive segregants more resistant than either parent and crossed them with elite romaine and crisphead types. We determined race 2 resistance is highly correlated with flowering time. We will complete evaluation of selected breeding lines in the next project plan for germplasm release. We continued our work to combine resistance to Verticillium Race 1 and Race 2. In 2019 and 2020, we reinoculated the Verticillium dahliae Race 1 disease nursery (0.5 acre) in Field C at the USDA-ARS station in Salinas. We evaluated commercial varieties, USDA breeding lines, and wild accessions for resistance to Race 1 in 2021, 2022, and 2023. Late season Race 1 plantings were abandoned due to high natural INSV infection. At maturity, few plants were remaining due to a combination of INSV, Verticillium, Fusarium, and Pythium. In the greenhouse, we evaluated thousands of individuals of RH12-3196 [Race 1 resistant]x (Salinas x PI 171674 [Race 2 resistant]) and genotyped with the Vr1 resistance-linked marker. Vr1-positive individuals were identified and were evaluated for resistance to Race 1, Race 2, and a mixed culture of the two in the greenhouse. We are finalizing germplasm evaluation for release in late FY23. In support of Sub-objectives 2A, 2B and 2C, we conducted recurrent selections to breed spinach for resistance to downy mildew, leafminers, and Linuron/Spin-Aid herbicides. Crosses were made among cultivars with different downy mildew, leafminer, and herbicide-resistant genes to combine their resistances. Progenies from 34 crosses, along with resistant and susceptible controls, were planted in replicated field trials at the USDA-ARS station in Salinas, California, to test their resistances. In our trials, two spinach breeding populations had 0 percent (%), and another five populations had under 10% downy mildew incidences, as compared to the susceptible control (‘Viroflay’) with 97% disease incidence. Our results show that the recurrent selection method was very effective at increasing downy mildew resistance in the spinach populations. Plants with resistances to downy mildew, leafminers, and Spin-Aid herbicide were selected and transplanted into our isolators to produce seeds for the next round of selection. In support of Sub-objectives 3A and 3B, we continued development of a 384-member Melon Core set under auspices of the CucCAP 2 project (Harnessing Genomic Resources For Disease Resistance And Management In Cucurbit Crops – Bringing The Tools To The Field). A collaborator/stakeholder produced S2 single seed descent progenies for 296 of the lines in the set. This company plans to advance 200 of them to the S3 as bulk sib increases. We recruited two additional collaborators/stakeholders to assist in advancing 40 and 25 lines to the S1 and S2 generations, respectively. The S3 generation when complete will be submitted as a set to NPGS GRIN (U.S. National Plant Germplasm System, Germplasm Resources Information Network) for distribution to melon researchers. The melon core set includes approximately 99% of the genetic variation in the U.S. melon collection and will be a valuable resource for genetic studies and germplasm screening for target traits important to the melon industry.

Accomplishments
1. Determined the inheritance of partial resistance to Verticillium dahliae race 2 in two resistant sources. Few chemical solutions are available to manage Verticillium in lettuce and new races are being identified on multiple crops throughout California. Resistant varieties are the best solution to control the disease. Determining how sources of resistance are inherited directs how best to incorporate this germplasm in a breeding program. ARS researchers in Salinas, California, have determined that the most efficient breeding strategy for Verticillium race 2 partial resistance is to make selections at late generations from a large population. Lettuce breeders will use this strategy to develop improved, resistant germplasm.

2. Identification and characterization of Yellow spot malady in lettuce. Lettuce is one of the most popular vegetable crops in the world. Most of the California lettuce production is concentrated in Monterey County where lettuce is grown across more than 40,000 ha with an annual value over $1 billion. In recent years, an unknown malady was observed on lettuce plants grown both in a greenhouse and field that affects visual appearance and marketability of whole heads, lettuce hearts, and fresh-cut lettuce. ARS researchers in Salinas, California, have provided the first comprehensive insight into the malady effect on lettuce growth and mineral composition of plants. ARS researchers identified lettuce cultivars with limited occurrence of Yellow spot malady (YSM) and mapped loci associated with resistance to YSM. Results of this study will be used in breeding programs to develop lettuce cultivars with improved resistance to YSM.

3. Field trials demonstrate resistance to Fusarium wilt in celery. Fusarium wilt is caused by the soil-borne pathogen Fusarium oxysporum f.sp. apii, race 2 and 4 are the primary pathogens affecting celery production in California. Genetic resistance through breeding efforts is the most effective control. ARS researchers at Salinas, California, with collaborators from University of California (UC) Division of Agriculture and Natural Resources, coordinated field trials in race 2 and race 4 infested soils to demonstrate new cultivars from private breeding companies with improved resistance to Fusarium wilt for stakeholders. ARS and UC researchers held field day events at both locations that were attended by growers, shippers, breeders, and public and private company researchers. The event generated considerable interest in promising new varieties from breeding companies. The data will be used to support release of new, resistant varieties from private breeding companies.

Review Publications
Bhattarai, G., Dotun, O., Mou, B., Correll, J., Shi, A. 2022. Mapping and selection of downy mildew resistance in spinach cv. Whale by low coverage whole genome sequencing. Frontiers in Plant Science. 13. Article 1012923. https://doi.org/10.3389/fpls.2022.1012923.
Mou, B. 2023. Green leaf, red leaf, and romaine lettuce breeding lines with resistance to leafminer, corky root, and downy mildew. HortScience. 58(4):436-441. https://doi.org/10.21273/HORTSCI17069-22.
Sthapit Kandel, J., Sandoya, G.V., Zhou, W., Read, Q.D., Mou, B., Simko, I. 2022. Identification of quantitative trait loci associated with bacterial leaf spot resistance in baby leaf lettuce. Plant Disease. 106(10):2583-2590. https://doi.org/10.1094/PDIS-09-21-2087-RE.
Sandoya, G.V., Rosenthal, E., Simko, I., Rodrigues-Porto, L.N., Wadlington, W.H., Bull, C.T., Carroll, A. 2022. Lettuce (Lactuca sativa L.) germplasm resistant to bacterial leaf spot caused by race 1 of Xanthomonas hortorum pv. vitians (Brown 1918) Moriniére et al. 2020. Journal of Plant Pathology. 104:993–1008. https://doi.org/10.1007/s42161-022-01123-0.
Simko, I., Puri, K.D., Dhar, N., Peng, H., Subbarao, K.V. 2022. Mapping quantitative trait loci for lettuce resistance to Verticillium dahliae race 3, plant development, and leaf color using an ultra-high-density bin map constructed from F2 progeny. Phytofrontiers. 2(3):257-267. https://doi.org/10.1094/PHYTOFR-11-21-0078-R.
Simko, I., Peng, H., Sthapit Kandel, J., Zhao, R.B. 2022. Genome-wide association mapping reveals genomic regions frequently associated with lettuce field resistance to downy mildew. Theoretical and Applied Genetics. 135:2009–2024. https://doi.org/10.1007/s00122-022-04090-3.
Peng, H., Zhao, R.B., Smith, R., Simko, I. 2022. Phenotypic and genetic analyses of yellow spot malady in lettuce. Scientia Horticulturae. 305. Article 111389. https://doi.org/10.1016/j.scienta.2022.111389.
Bhattarai, G., Shi, A., Mou, B., Correll, J. 2022. Resequencing worldwide spinach germplasm for identification of field resistance QTLs to downy mildew and assessment of genomic selection methods. Horticulture Research. 9. Article uhac205. https://doi.org/10.1093/hr/uhac205.
Mou, B., Eriksen, R.L. 2022. Vegetable cultivar descriptions for North America List 28. HortScience. 57(8):949-1040. https://doi.org/10.21273/HORTSCI.57.8.949.
Brandl, M., Mammel, M., Simko, I., Richter, T., Gebru, S., Leonard, S. 2023. Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce. Food Microbiology. 113. Article 104260. https://doi.org/10.1016/j.fm.2023.104260.