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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #434549
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

2022 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 in Salinas, California, continued to make crosses, selections, and seed increases to breed for resistances to leafminers, corky root, and yellow spot, nutritional improvement, appearance, 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 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 are in the process of public release. In support of Sub-objective 1B, research continued on mapping major Quantitative Trait Loci (QTL) for resistance to downy mildew (DM) and development of lettuce breeding lines with 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. Results were published in two peer-reviewed papers. In support of Sub-objective 1C, research shifted to understanding reports of “new races” in commercial fields. Samples from two fields yielded distinctly different isolates that appear to represent distinct race types based on an informally defined international race differential set. Discussions with ARS and university plant pathologists are leading to coordinated efforts to biological and molecular characterization of these and other isolates and a clear direction for resistance breeding. In support of Sub-objective 1E, research was performed to identify and map major QTL for resistance to lettuce drop and to develop breeding lines of romaine lettuce with improved resistance to lettuce drop. A linkage map of the mapping population was developed using SNP-based markers. Phenotypic data for lettuce resistance to sclerotia wilt were collected from one field trial. The 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 continued on development of tools for automatic phenotyping of lettuce deterioration. Phenotypic data of deterioration were collected from over 1,000 samples of fresh-cut lettuce. Results were published in two peer-reviewed papers. In support of Sub-objective 1H, we planted a replicated field trial of 174 breeding lines segregating for tipburn resistance, parent lines, and check varieties. The trial was hit hard by downy mildew and impatiens necrotic spot virus (INSV) and nearly every plot exhibited disease symptoms. Seed of backcross progeny was increased and generation advanced in the greenhouse. In support of Sub-objective 1I, we continued efforts to optimize high-throughput INSV phenotyping in the field and greenhouse. In early FY22, we harvested early- and late-season field trials to capture variation in disease pressure throughout the season. Both trials were a Randomized Complete Block Design (RCBD) with three replications. The late-season trial captures the peak thrips pressure in the area as previously determined by ARS scientists. We flagged 10 plants per plot for weekly individual evaluation. At 6, 7, 8, 9, and 10 weeks after planting, we assigned each of the 10 plants a disease severity rating (0-5 scale). The weekly mean INSV severity scores were combined into a single value using the Area Under the Disease Progress Staircase (AUDPS). AUDPS values range from 0 (all plants had a severity of 0 for all five weeks) to 25 (all plants had a severity of 5 for all five weeks). AUDPS values were significantly higher in the August planting, confirming variable disease pressure throughout the growing season. The same material was tested under greenhouse conditions. 5-week old seedlings (10 per line) were manually inoculated with INSV sap and exposed to viruliferous thrips. Each plant was rated for INSV severity (0-5) at 2, 3, 4, 5, and 6 weeks after inoculation. Rating times for field and greenhouse were based on when first symptoms appeared on the susceptible check. INSV severity scores were combined into a single value using the AUDPS calculation. AUDPS INSV severity values in the greenhouse test were significantly higher than both the early- and late-season field trials. This suggests the manual inoculation plus viruliferous thrips transmission might not be reflective of field conditions. Early- and late-season trials were planted again in June and August 2022. In support of Sub-objective 1J, we continued our work to combine resistance to Verticillium Race 1 and Race 2 and develop Verticillium Race 2 resistant breeding lines. 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. 2021 was our first opportunity to evaluate germplasm in the field. We planted a uniformity trial in mid-May to determine disease pressure throughout the field and how best to block experiments to account for that variation. The East end of the field had higher disease severity values than the West end. In August we planted our first field trial for breeding line evaluation. Unfortunately, the trial was hit hard by INSV. At maturity, few plants were remaining due to a combination of INSV, Verticillium, Fusarium, and Pythium. Trials were harvested and data analyzed in early FY22. We adjusted our planting dates in 2022 and trials were planted in April and May to avoid heavy INSV pressure in late-season experiments. In the greenhouse, we collected leaf tissue from 1,592 F2 individuals of RH12-3196 x (Salinas x PI 171674) and genotyped all with the Vr1 resistance-linked marker. This population combines Race 1 resistance (Vr1) from RH12-3196 with Race 2 resistance from PI 171674 in a crisphead type (Salinas). 1,117 Vr1-positive individuals were identified and were evaluated for resistance to Race 2 in the greenhouse. We evaluated 3192 individuals of 258 F3 families of 11-G99-1-1 x PI 171674 for resistance to V. dahlia 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. Analysis of results is underway. In support of Sub-objectives 2A, 2B and 2C, we conducted recurrent selections to breed spinach for resistance to downy mildew, leafminers, and Spin-Aid herbicide. In our trials, two spinach breeding populations had 0%, 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-objective 3B, we completed production of four F2:3 populations from crosses of whitefly-susceptible western shipping type cantaloupe cultivar Top Mark with three putative sources of whitefly resistance (PI 122847, PI 313970, PI 414723 and TGR 1551).


Accomplishments
1. Lettuce varieties with resistance to leafminers, corky root, and downy mildew. Leafminer, corky root, and downy mildew are major pests and diseases of lettuce. The most economic means of control are through the use of resistant cultivars. ARS researchers in Salinas, California, developed and released two green leaf, a red leaf, and two romaine lettuce varieties with resistance to these pests and diseases. The varieties may be used for commercial production, and are suitable for use as sources of resistance in the development of new lettuce cultivars by other public and private breeders.

2. Seasonal effects connected to E. coli outbreaks in fresh cut lettuce stored at modified atmosphere packaging. Escherichia coli (E. coli) O157:H7 infection outbreaks connected to fresh cut lettuce stored in modified atmosphere packaging (MAP) are more frequently associated with crops harvested at the end of the growing season than with crops harvested at the beginning or during growing season. ARS researchers in Albany and Salinas, California, together with colleagues from the U.S. Food and Drug Administration in Maryland, showed that greater postharvest deterioration in MAP in the fall lettuce was associated with improved pathogen survival. The research team also demonstrated that the bacterial community present on bagged lettuce differed by season, lettuce deterioration state, and whether survival of E. coli on the lettuce was high or low. This suggests a potential for using the microbiome as an indicator of the microbial quality of fresh-cut bagged lettuce.


Review Publications
Mamo, B., Eriksen, R.L., Adhikari, N.D., Hayes, R.J., Mou, B., Simko, I. 2021. Epidemiological characterization of lettuce drop (Sclerotinia spp.) and biophysical features of the host identify soft stem as a susceptibility factor. Phytofrontiers. 1(3):182-204. https://doi.org/10.1094/PHYTOFR-12-20-0040-R.
 Simko, I. 2021. IdeTo: Spreadsheets for calculation and analysis of area under the disease progress over time data. Phytofrontiers. 1(3):244-247. https://doi.org/10.1094/PHYTOFR-11-20-0033-A.
 An, G., Simko, I., Chen, J., Yu, C., Lavelle, D., Zhang, W., Michelmore, R., Kuang, H. 2021. Hypersensitivity to triforine in lettuce is triggered by a TNL gene through the disease resistance pathway. Plant Biotechnology Journal. 19(11):2144-2146. https://doi.org/10.1111/pbi.13679.
 Guo, J., Dong, L., Kandel, S.L., Jiao, Y., Shi, L., Yang, Y., Shi, A., Mou, B. 2022. Transcriptomic and metabolomic analysis provides insights into the fruit quality and yield improvement in tomato under soilless substrate-based cultivation. Agronomy. 12(4). Article 923. https://doi.org/10.3390/agronomy12040923.
 Kandel, S.L., Henry, P.M., Goldman, P.H., Mou, B., Klosterman, S.J. 2022. Composition of the microbiomes from spinach seeds infested or noninfested with Peronospora effusa or Verticillium dahliae. Phytobiomes Journal. 6(2):169-180. https://doi.org/10.1094/PBIOMES-05-21-0034-R.
 Kandel, S.L., Anchieta, A.G., Shi, A., Mou, B., Klosterman, S.J. 2022. Crustacean meal elicits expression of growth and defense-related genes in roots of lettuce and tomato. Phytofrontiers. 2(1):10-20. https://doi.org/10.1094/PHYTOFR-03-21-0017-R.
 Bhattarai, G., Yang, W., Shi, A., Feng, C., Dhillon, B., Correll, J.C., Mou, B. 2021. High resolution mapping and candidate gene identification of downy mildew race 16 resistance in spinach. BMC Genomics. 22. Article 478. https://doi.org/10.1186/s12864-021-07788-8.
 Chen, J., Zhang, D., Huang, J., Li, R., Wang, D., Song, J., Puri, K.D., Lin, Y., Kong, Z., Tong, B., Li, J., Huang, Y., Simko, I., Klosterman, S.J., Dai, X., Subbarao, K.V. 2021. Dynamics of Verticillium dahliae race 1 population under managed agricultural ecosystems. BMC Biology. 19. Article 131. https://doi.org/10.1186/s12915-021-01061-w.
 Sandoya, G.V., Truco, M.J., Bertier, L.D., Subbarao, K.V., Simko, I., Hayes, R.J., Michelmore, R.W. 2021. Genetics of partial resistance against Verticillium dahliae race 2 in wild and cultivated lettuce. Phytopathology. 111(5):842-849. https://doi.org/10.1094/PHYTO-09-20-0396-R.
 Peng, H., Luo, Y., Teng, Z., Zhou, B., Bornhorst, E.R., Fonseca, J.M., Simko, I. 2021. Phenotypic characterization and inheritance of enzymatic browning on cut surfaces of stems and leaf ribs of romaine lettuce. Postharvest Biology and Technology. 181. Article 111653. https://doi.org/10.1016/j.postharvbio.2021.111653.
 Rosental, L., Still, D.W., You, Y., Hayes, R.J., Simko, I. 2021. Mapping and identification of genetic loci affecting earliness of bolting and flowering in lettuce. Theoretical and Applied Genetics. 134:3319-3337. https://doi.org/10.1007/s00122-021-03898-9.
 Simko, I., Jia, M., Venkatesh, J., Kang, B., Weng, Y., Barcaccia, G., Lanteri, S., Bhattarai, G., Foolad, M.R. 2021. Genomics and marker-assisted improvement of vegetable crops. Critical Reviews in Plant Sciences. 40(4):303-365. https://doi.org/10.1080/07352689.2021.1941605.
 Liu, Z., Sun, J., Teng, Z., Luo, Y., Yu, L., Simko, I., Chen, P. 2021. Identification of marker compounds for predicting browning of fresh-cut lettuce using untargeted UHPLC-HRMS metabolomics. Postharvest Biology and Technology. 180.Article 111626. https://doi.org/10.1016/j.postharvbio.2021.111626.
 Leonard, S., Simko, I., Mammel, M., Richter, T., Brandl, M. 2021. Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California. Environmental Microbiome. 16. Article 25. https://doi.org/10.1186/s40793-021-00393-y.
 Kumar, P., Eriksen, R.L., Simko, I., Shi, A., Mou, B. 2022. Insights into nitrogen metabolism in the wild and cultivated lettuce as revealed by transcriptome and weighted gene co-expression network analysis. Scientific Reports. 12. Article 9852. https://doi.org/10.1038/s41598-022-13954-z.
 Shi, A., Bhattarai, G., Xiong, H., Avila, C., Feng, C., Liu, B., Joshi, V., Stein, L., Mou, B., du Toit, L., Correll, J.C. 2022. Genome-wide association study and genomic prediction of white rust resistance in USDA GRIN spinach germplasm. Horticulture Research. 9. Article uhac069. https://doi.org/10.1093/hr/uhac069.