Research Project: Trait Discovery, Genetics, and Enhancement of Allium, Cucumis, and Daucus Germplasm

Location: Vegetable Crops Research

2023 Annual Report

Objectives
Objective 1: Phenotype, map, and identify traits of critical importance for vegetable growers, seed companies, and consumers in elite populations and in diverse genetic resources of Allium, Cucumis, and Daucus. Objective 2: Develop and release enhanced germplasm of Allium, Cucumis, and Daucus with superior traits. Objective 3: Determine the genetic bases and molecular processes for biotic and abiotic resistance, growth and productivity, nutritional value, and flavor characteristics in Allium, Cucumis, and Daucus. Objective 4: Develop informational resources and tools to evaluate phenotypic and genotypic data from Allium, Cucumis, and Daucus breeding and genetic research.

Approach
The long-term potential for improving a crop is only as great as the breadth of diversity that breeders utilize. Objective 1: Identify unique phenotypic variation in carrot, onion, and cucumber germplasm collections and breeding stocks and genetically map key traits to improve nutritional and processing quality, disease resistance, stress tolerance, and yield of Allium, Cucumis, and Daucus vegetables, characterize observed variation and initiate genetic incorporation of these phenotypes into elite germplasm. Objective 2: Incorporate valuable traits and release elite germplasm and genetic stocks using marker-assisted selection and provide stakeholders with germplasm and databases including maps. Dense genetic maps are useful to improve the efficiency of crop improvement. We will identify unique phenotypes in elite onion, cucumber, and carrot germplasm to construct genetic maps for marker-facilitated selection of major horticultural traits. Objective 3: Develop populations to determine the patterns of inheritance of unique phenotypic variation and develop molecular markers for traits in germplasm collections and breeding stocks to improve nutritional and processing quality, disease resistance, stress tolerance, and yield of Allium, Cucumis, and Daucus vegetables, phenotype observed variation among individuals in populations, and develop genetic models to explain observed genetic patterns. Information on trait genetics from germplasm evaluation and genetic analysis is useful and sets the stage for developing genetic and breeding stocks, and for establishing information resources for stakeholders. Objective 4: Summarize and catalog phenotypic, genotypic, and molecular data collected and develop accessible and searchable databases.

Progress Report
This is the final report for this project which terminated in March 2023. See the report for the replacement project, 5090-21000-073-000D, “Trait Discovery, Genetics, Genomics, and Enhancement of Cucumis, Daucus, and Allium Germplasm” for additional information. (Objective 1) Many cucumber lines were phenotyped in greenhouse, growth chamber and field trials to identify various horticulturally important traits including fruit size and shape, hypocotyl length, flowering time, parthenocarpy fruit setting, abiotic stress tolerance (low temperature germination), and disease resistances (downy mildew, powdery mildew, angular leaf spot, and anthracnose). A panel of 399 cucumber lines were established genome-wide association study (GWAS). Inbred lines for lines in the GWAS panel were developed by continuous self-pollination. This panel was subjected for multiple-year, multiple environment phenotyping focusing on downy and powdery mildew resistances, and plant architecture and yield-related traits. Multiple new cucumber mapping populations were developed for framework or fine genetic mapping of genes or quantitative trait loci (QTL) for fruit size/shape, fruit taste quality, and disease resistances. Insects and microbial diseases can significantly reduce the commercial value of the onion crop, but in a diverse collection of onion varieties held in the USDA germplasm collection, some varieties exhibit minimal damage due to these insects and diseases. Seed was produced from onions selected over years for less damage under natural pressure from the insect referred to as thrips, which not only damages the leaves of onion plants, but also vectors viral diseases that significantly reduce the productivity of the onion crop. Variation in the amounts and types of epicuticular waxes on the surface of onion leaves was found to significantly influence the damage caused by thrips and in an analysis of onion breeding stocks, a single gene was demonstrated to control much of the variation in wax composition. Beyond the insect damage to the onion crop due to thrips, two microbial diseases called pink root and Fusarium basal rot also significantly reduce onion productivity and postharvest storage quality. Carrot provides 12-13% of the dietary vitamin A for U.S. consumers and new breeding populations with higher nutritional value. Novel colors were identified and advanced to increase seed production and expand testing. Experimental hybrid carrots from the USDA program performed very well in trial comparisons with commercial cultivars and seed released by the USDA program to the U.S. seed industry is used by them to develop the majority of the current U.S. crop. USDA carrot breeding selections with very good flavor and nematode resistance were among those with superior field performance, and unique, superior nematode resistance derived from USDA carrot germplasm contributes to reduced use of pesticides for production of the U.S. crop. Carrot populations selected for the expanding U.S. organic carrot industry were advanced in the breeding program. (Objective 2) The abovementioned cucumber work resulted in many inbred lines, introgression lines, near-isogenic lines which may be of value to plant breeders or cucurbit researchers. These germplasm lines include the GWAS panel of 399 inbred lines from the core collection will be made publicly available. Given the significant damage that thrips inflict on onion due to their feeding and to the viruses they carry, the development of DNA markers were developed to track this gene to develop thrips resistant onion cultivars. Onion selections with high levels of resistance to pink root and Fusarium basal rot were used to track the incorporation of genetic resistance to these two diseases and a single gene was found to confer significant resistance to pink root, and three genes were found to control resistance to Fusarium. Carrot genetic mapping populations and diverse germplasm were phenotyped for nematode and Alternaria leaf blight resistance, top size, flavor, and root pigment content, and molecular genetic markers were developed to incorporate these traits into new breeding stocks. In a subset of these genetic stocks, variation in the interaction between carrots and native beneficial soil microbes was observed, suggesting that these plant-microbe interactions influence plant growth and resistance to Alternaria leaf blight, a major foliar disease of carrots. Comparisons were made for these interactions with carrots grown under conventional farming practices and under organic farming practices, and these contrasting production practices revealed additional differences in plant-microbe interactions. The carrot genome sequence was completed in a collaborative project led by ARS researchers in Madison, Wisconsin, along with collaborators at the University of California-Davis. The carrot genome has been used to provide molecular genetic markers to better understand angiosperm evolution, the genetic and molecular basis of orange and purple carrot color, and the identity of genes associated with the domestication of carrot. Orange and purple carrots from a collection of approximately 700 wild and cultivated carrot germplasm accessions were evaluated for variation in the orange carotenoid and purple anthocyanin tap root pigments. Until recently, two genes, Y and Y2, had been identified that account for the dramatic shift from yellow to orange carrots. But within the last decade two more genes, Or and CH, were found to also account for the orange color typical of carrots. A wide range of stress and disease tolerance, vigor, and consumer quality was also observed in this germplasm collection. (Objective 3) The whole genomes of the cucumber GWAS panel were genotyped with genotyping-by-sequencing (GBS) as well as whole genome resequencing. The genetic diversity and population structure of the GWAS population was evaluated. Many segregating populations (F2, F2:3 and recombinant inbred lines) were developed for target traits, and linkage maps were developed using genotyping-by-sequencing, and the DArTagging SNP genotyping technologies. QTL analysis, linkage analysis and association analysis were performed in these populations. Genes and QTLs for many traits were identified to understand the genetic basis of these traits. Several genes or disease resistance QTK were fine mapped or cloned, which helped elucidating the underlying molecular mechanisms regulating those traits New red and yellow onion breeding stocks were developed with resistance to both pink root and Fusarium basal rot. In addition to these breeding stocks, one onion hybrid and seven other onion breeding populations with novel bulb color, mild flavor, and very long storage ability, were released to vegetable breeders and researchers. Carrot inbred breeding stocks and breeding pools incorporating newly characterized genes for improved productivity, nematode and Alternaria leaf blight resistance, flavor, color and nutritional value was initiated and advanced to release for use by carrot breeding programs and evaluation by researchers. (Objective 4) This project also generate wealthy information on cucumber genome sequences for the core collection, molecular markers, gene sequences for target traits, phenotypic data for the GWAS panel genetic maps, information for genes and QTL. All these resources are being made available to the public through publications and publicly accessible databases Plant germplasm evaluation and plant breeding and genetics programs for crops including carrot generate large datasets to characterize crop traits and their genetic makeup. These datasets are often grouped into databases by the crop, or family of related crops, that have been characterized. In this project a database called CarrotOmics was developed to provide a resource for collecting and organizing genetic and agricultural data for carrot and related crops. The images and genomic data collected constitutes a database that is of vital importance and interest to plant researchers, crop breeders in public and private sector, crop production and processing industries, and consumers. The carrot breeding stocks, genetic information, and CarrotOmics database provide a valuable foundation for ongoing research.

Accomplishments
1. Map-based cloning of a new gene for downy mildew resistance in cucumber. The gene dm5.3 confers high-level resistance against downy mildew (DM), which is a severe disease in cucumber production, but the underlying resistance mechanism is unknown. ARS scientists in Madison, Wisconsin, conducted fine genetic mapping of dm5.3 to identify the gene and obtain clues to the mechanism. Extensive phenotyping and fine mapping narrowed dm5.3 into a 144 kilobase region. Multiple lines of evidence identified the ‘sigma factor binding protein 1’ (CsSIB1), a member of the VQ motif-containing protein family as the gene responsible. A single base pair mutation in CsSIB1 was likely the cause of dm5.3-conferred DM resistance. In response to DM pathogen inoculation, CsSIB1 exhibited higher expression in the resistant than in the susceptible plants. Identification of the gene will lead to an improved understanding of the molecular mechanisms of DM resistance in cucumber and other crop plants. This work also provides molecular markers that could be used for marker-assisted breeding of DM resistance in cucumber breeding.

2. Development of a database for carrots and related crops. In crop improvement programs large datasets to characterize crop traits and their genetic makeup are generated. These datasets, known as databases, are often grouped by the crop, or family of related crops, that have been characterized. With recent sequencing of the carrot genome, large datasets are rapidly being generated, so ARS researchers in Madison, Wisconsin, developed a database called CarrotOmics to provide a resource for collecting and organizing agricultural and genetic data for carrot and related crops. The images and genomic data collected constitutes a database that is of great interest to plant researchers, crop breeders in public and private sector, crop production and processing industries, and consumers since the carrot breeding stocks and genetic information in the CarrotOmics database provides a valuable foundation for ongoing research.

Review Publications
Trivino, N., Rodriguez-Sanchez, A., Filley, T., Camberato, J., Colley, M., Simon, P.W., Hoagland, L. 2023. Carrot genotypes differentially alter soil microbial communities and scavenge nitrogen from organic materials in soil. Frontiers in Plant Science. Article (the author) 2023. https://doi.org/10.1007/s11104-023-05892-0.
Che, G., Pan, Y., Liu, X., Li, M., Zhao, J., Yan, S., Yuting, H., Wang, Z., Cheng, Z., Song, W., Zhou, Z., Wu, T., Weng, Y., Zhang, X. 2022. Natural Variation in CRABS CLAW contributes to fruit length divergence in cucumber. The Plant Cell. 35(2). https://doi.org/10.1093/plcell/koac335.
Nijabat, A., Manzoor, S., Faiz, S., Naveed, N., Bolton, A., Khan, B., Ali, A., Simon, P.W. 2023. Variation in seed germination and amylase activity of diverse carrot [daucus carota (L.)] germplasm under simulated drought stress. HortScience. 58(2):205-214. https://doi.org/10.21273/HORTSCI16806-22.
Rolling, W.R., Senalik, D.A., Iorizzo, M., Ellison, S., Van Deynze, A., Simon, P.W. 2022. CarrotOmics: a genetics and comparative genomics database for carrot (Daucus carota). Horticulture Research. 2022, 1-8. https://doi.org/10.1093/database/baac079.
Chintha, P., Sarkar, D., Pecota, K., Dogramaci, M., Shetty, K. 2021. Improving phenolic bioactive-linked functional qualities of sweet potatoes using beneficial lactic acid bacteria-based biotransformation strategy. Horticulturae. 7(10). Article e367. https://doi.org/10.3390/horticulturae7100367.
Brainard, S., Ellison, S., Simon, P.W., Dawson, J., Goldman, I. 2021. Genetic characterization of carrot root shape and size using genome-wide association analysis and genomic-estimated breeding values.. Theoretical and Applied Genetics. 135:605–622. https://doi.org/10.1007/s00122-021-03988-8.
Zhai, X., Wu, H., Wang, Y., Zhang, Z., Shan, L., Zhao, X., Wang, R., Liu, C., Weng, Y., Wang, Y., Liu, X., Ren, H. 2022. The fruit glossiness locus, dull fruit (D) encodes a C2H2 type zinc finger transcription factor CsDULL in cucumber (Cucumis sativus L.). Horticulture Research. 2022, 9: uhac146. https://doi.org/10.1093/hr/uhac146.
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.K., Ando, K., Mccreight, J.D., Schaffer, A., Burger, J., Tadmor, Y., Katzir, N., Tang, Z., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, P.W., 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. (2019): Vol. 4747(D1):D1128-D1136.. https://doi.org/10.1093/nar/gky944.
Tan, J., Wang, Y., Dymerski, R.D., Wu, Z., Weng, Y. 2022. Sigma factor binding protein 1 (CsSIB1) is a putative candidate of the major-effect QTL dm5.3 for downy mildew resistance in cucumber (Cucumis sativus). Theoretical and Applied Genetics. 135:4197–4215. https://doi.org/10.1007/s00122-022-04212-x.
Chen, F., Yong, J., Zhang, G., Liu, M., Wang, Q., Zhong, H., Pan, Y., Chen, P., Weng, Y., Li, Y. 2023. LTR retrotransposon insertion inside CsERECTA for a LRR receptor-like Serine/threonine-protein kinase results in compact (cp) plant architecture in cucumber. Journal of Theoretical and Applied Genetics. Article number 31. https://doi.org/10.1007/s00122-023-04273-6.
Feng, Z., Sun, L., Dong, M., Fan, S., Xi, K., Wang, W., Song, L., Weng, Y., Liu, X., Ren, H. 2023. Novel players in organogenesis of and flavonoid biosynthesis in cucumber glandular trichomes. Plant Physiology. 192 (4), 2723-2736. https://doi.org/10.1093/plphys/kiad236.
Liu, H., Zhao, J., Chen, F., Wu, Z., Tan, J., Nguyen, N., Cheng, Z., Weng, Y. 2023. Improving Agrobacterium tumefaciens-mediated genetic transformation for gene function studies and mutagenesis in cucumber (Cucumis sativus). Genes. 14(3), 601. https://doi.org/10.3390/genes14030601.
Zhao, J., Bo, K., Pan, Y., Li, Y., Yu, D., Li, C., Chang, J., Wu, S., Wang, Z., Zhang, X., Gu, X., Weng, Y. 2022. Phytochrome-interacting factor PIF3 integrates phytochrome B and UVB signaling pathways to regulate gibberellin- and auxin-dependent growth in cucumber hypocotyls. Journal of Experimental Botany. 74(15):4520-4539. https://doi.org/10.1093/plcell/koac335.
Perez, M.B., Carvajal, S., Beretta, V., Bannoud, F., Fangio, M.F., Berli, F., Fontana, A., Salomon, M.V., Gozalez, R., Valerga, L., Altamirano, J.C., Yildiz, M., Iorizzo, M., Simon, P.W., Cavagnaro, P.C. 2023. Characterization of purple carrot germplasm for antioxidant capacity and root concentration of anthocyanins, phenolics, and carotenoids. Plants. 12(9). Article 1796. https://doi.org/10.3390/plants12091796.