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

2021 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 at Salinas, California, continued to make crosses, selections, and seed increases to breed for resistance to leafminers, corky root, yellow spot, tolerance to herbicides, 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. 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 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 the F4 filial generation were produced from 27 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. 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. Seed of nine F4 filial generation lines were produced in a greenhouse. 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 852 samples of fresh-cut lettuce. Phenotyping of 500 accessions with FluorCam has been performed. Sub-objective 1H is to develop romaine breeding lines with reduced incidence of tipburn. 244 breeding lines were grown in a replicated field experiment and evaluated for incidence of tipburn and horticultural traits. 30 F1 populations segregating for tipburn resistance are growing in the greenhouse for generation advance and seed increase. Seeds of 64 lines from Green Towers x Salinas that were identified in a 2019 field trial with low incidence of tipburn were increased in the greenhouse. We also advanced in generation 30 backcross lines between Green Towers x Salinas selected progeny and elite romaine type varieties. Sub-objective 1I is to identify lettuce germplasm with resistance to impatiens necrotic spot virus (INSV), a thrips-vectored orthotospovirus that causes economic damage on lettuce in coastal California, associated with large populations of its thrips vector. In a late season planting in 2020, we evaluated 36 varieties and 76 breeding lines in a randomized experiment with four replications, with INSV incidence (proportion of plants per plot showing symptoms) measured weekly, followed by a disease severity rating (0-5 scale) at harvest with leaf tissue evaluated by enzyme-linked immunoassay (ELISA) to determine presence or absence of INSV. Red leaf lines were determined to have lower INSV incidence than green leaf lines under lower disease pressure (average incidence below 20%) but not under higher disease pressure, and INSV severity ratings were highly correlated (81.8%) with INSV incidence ratings, but ELISA results did not clearly correlate with either severity or incidence. Only one line was identified that remained uninfected based on ELISA. Field results were validated in greenhouse experiments with manual inoculation and viruliferous thrips transmission with greenhouse results closely matching those from the field. Mapping populations with identified sources of resistance are in development. Sub-objective 1J is to identify higher levels of resistance to Verticillium dahliae race 2 and develop resistant iceberg lettuce. We evaluated 424 F3 individuals for resistance to V. dahlia Race 2 under growth room, controlled conditions. 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. No lines were significantly more resistant than either parent. 21 lines were selected with low vascular symptoms, low bolting incidence, and a romaine-like type.

Accomplishments
1. Predictive modeling of a leaf conceptual midpoint quasi-color (CMQ) using an artificial neural network. Lettuce is one of the most valuable fresh vegetables and is in the top 10 most valuable crops in the United States, with an annual farm-gate value of over $2.5 billion. The color of lettuce leaves may show considerable difference between the two leaf surfaces. ARS scientists in Salinas, California, have developed a method to predict a leaf surface color from the content of pigments. The color predicted from the content of chlorophylls and anthocyanins by means of an artificial neural network (ANN), matched well with the color determined empirically. The model predicted a substantially duller color for leaves with chlorophylls and anthocyanins present together, particularly when both pigments were present at very high levels. Leaf color is important to the lettuce industry. This approach could be used to model leaf colors in environments where it is not possible to observe both leaf surfaces, such as the color of the lower surface of prostrate leaves grown in controlled-environment agriculture (indoor farms).

2. QTL mapping of host plant resistance to cucurbit yellow stunting disorder virus in melon. The western U.S. melon (cantaloupe) industry was hard hit by a combination of sweet potato whitefly and cucurbit yellow stunting disorder virus (CYSDV) in 2007, virtually eliminating fall melon production in the lower desert areas. These problems recently emerged in the major summer melon production area as well. High-level, Mendelian resistance to the virus was identified in several vegetable type melons from India and Africa by ARS scientists at Salinas, California. Two quantitative trait loci (QTL) were identified in melon PI 313970, and one or both markers were present in six of 10 putative melon CYSDV resistance sources. Markers flanking the QTL were developed and can be used in marker-assisted breeding of CYSDV-resistant melons. These results will facilitate development of CYSDV-resistant melon cultivars for the lower desert areas of the United States.

Review Publications
Bhattarai, G., Shi, A., Feng, C., Dhillon, B., Mou, B., Correll, J.C. 2020. Genome wide association studies in multiple spinach breeding populations refine downy mildew Race 13 resistance genes. Frontiers in Plant Science. 11. https://doi.org/10.3389/fpls.2020.563187.
Lafta, A., Sandoya, G., Mou, B. 2020. Genetic variation and genotype by environment interaction for heat tolerance in crisphead lettuce. HortScience. 56(2):126-135. https://doi.org/10.21273/HORTSCI15209-20.
Parra, L., Simko, I., Michelmore, R.W. 2021. Identification of major quantitative trait loci controlling field resistance to downy mildew in cultivated lettuce (Lactuca sativa). Phytopathology. 111(3):541-547. https://doi.org/10.1094/PHYTO-08-20-0367-R.
Kumar, P., Eriksen, R.L., Simko, I., Mou, B. 2021. Molecular mapping of water-stress responsive genomic loci in lettuce (Lactuca spp.) using kinetics chlorophyll fluorescence, hyperspectral imaging and machine learning. Frontiers in Genetics. 12. Article 634554. https://doi.org/10.3389/fgene.2021.634554.
Park, S., Kumar, P., Shi, A., Mou, B. 2021. Population genetics and genome-wide association studies provide insights into the influence of selective breeding on genetic variation in lettuce. The Plant Genome. 14(2). Article e20086. https://doi.org/10.1002/tpg2.20086.
Tamang, P., Ando, K., Wintermantel, W.M., McCreight, J.D. 2021. QTL mapping of Cucurbit yellow stunting disorder virus resistance in melon accession PI 313970. HortScience. 56(4):424-430. https://doi.org/10.21273/HORTSCI15495-20.
Nguyen, C.D., Li, J., Mou, B., Gong, H., Huo, H. 2021. A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce. Vegetable Research. 1. Article 4. https://doi.org/10.48130/VR-2021-0004.
Zhou, W.Q., Zhou, Y.Q., He, C.Y., Mou, B., Zhou, W. 2020. Over-expression of Oshox4 enhances drought and salinity tolerance in rice. Russian Journal of Plant Physiology. 67:1152-1162. https://doi.org/10.1134/S1021443720060205.
Clark, K.J., Feng, C., Dhillon, B., Kandel, S.L., Poudel, B., Mou, B., Klosterman, S.J., Correll, J.C. 2020. Evaluation of spinach cultivars for downy mildew resistance in Yuma, AZ 2020. Plant Disease Management Reports. 14. Article V146.
Ravelombola, W., Shi, A., Chen, S., Xiong, H., Yang, Y., Cui, Q., Olaoye, D., Mou, B. 2020. Evaluation of cowpea for drought tolerance at seedling stage. Euphytica. 216. Article 123. https://doi.org/10.1007/s10681-020-02660-4.
Adhikari, N.D., Eriksen, R.L., Shi, A., Mou, B. 2020. Proteomics analysis indicates greater abundance of proteins involved in major metabolic pathways in Lactuca sativa cv. Salinas than Lactuca serriola accession US96UC23. Proteomics. 20(19-20). Article 1900420. https://doi.org/10.1002/pmic.201900420.
Eriksen, R.L., Adhikari, N.D., Mou, B. 2020. Comparative photosynthesis physiology of cultivated and wild lettuce under control and low-water stress. Crop Science. 60(5):2511–2526. https://doi.org/10.1002/csc2.20184.
Kandel, S.L., Hulse-Kemp, A.M., Stoffel, K., Koike, S.T., Shi, A., Mou, B., Van Deynze, A., Klosterman, S.J. 2020. Transcriptional analyses of differential cultivars during resistant and susceptible interactions with Peronospora effusa, the causal agent of spinach downy mildew. Scientific Reports. 10. Article 6719. https://doi.org/10.1038/s41598-020-63668-3.
Chen, J.-Y., Zhang, D.-D., Huang, J.-Q., Wang, D., Hao, S.-J., Li, R., Puri, K.D., Yang, L., Tong, B.-Z., Xiong, K.-X., Simko, I., Klosterman, S.J., Subbarao, K.V., Dai, X.-F. 2020. Genome sequence of Verticillium dahliae race 1 isolate VdLs.16 from lettuce. Molecular Plant-Microbe Interactions. 33(11):1265-1269. https://doi.org/10.1094/MPMI-04-20-0103-A.
Macias-Gonzalez, M., Truco, M.J., Smith, R., Cahn, M.D., Simko, I., Hayes, R.J., Michelmore, R.W. 2021. Genetics of robustness under nitrogen- and water-deficient conditions in field-grown lettuce. Crop Science. 61(3):1582-1619. https://doi.org/10.1002/csc2.20380.
Peng, H., Sthapit Kandel, J., Michelmore, R.W., Simko, I. 2020. Identification of factors affecting the deterioration rate of fresh-cut lettuce in modified atmosphere packaging. Food and Bioprocess Technology. 13:1997–2011. https://doi.org/10.1007/s11947-020-02538-2.
Zhou, W., Li, Z., Zhang, J., Mou, B., Zhou, W. 2021. The OsIME4 gene identified as a key to meiosis initiation by RNA in situ hybridization. Plant Biology. 23(5):861-873. https://doi.org/10.1111/plb.13274.