Research Project: Potato Germplasm Development for Improved Sustainability, Disease Resistance, Nutrition, and Quality

Location: Temperate Tree Fruit and Vegetable Research

2024 Annual Report

Objectives
A priority need for agricultural research in the coming years is to help ensure food security despite challenges such as an increasing population, climate uncertainty, rising input costs, and loss of arable land. Another need for potato is research that helps the industry adapt to evolving consumer preferences because consumers are increasingly prioritizing sustainability and nutritional value when making their food purchasing decisions. Our research will address these needs using both pre-breeding and breeding approaches to identify or develop potato germplasm with better disease and pest resistance, nutritional value, sustainability, and product quality. Our project has three interrelated objectives, all of which are ultimately intended to facilitate the development of superior new potato cultivars. OBJECTIVE 1: Utilize high-throughput phenotyping, molecular breeding strategies, and genomic prediction to characterize, breed, and release potato germplasm with improved traits, especially those related to disease and pest resistance, sustainability, and increased amounts of phytonutrients. Sub-objective 1A: Develop and deploy high-throughput phenotyping workflows to quantify foliar and tuber characteristics of individual clones within potato breeding populations. Sub-objective 1B: Generate and characterize multi-parent breeding populations that segregate for dominantly inherited, large-effect, pathogen resistance alleles. Sub-objective 1C: Screen cultivars, landraces, and wild species for resistance to soil-borne pathogens and develop self-compatible, diploid introgression populations. Sub-objective 1D: Develop new baby potato lines and characterize the genetics of traits important for a baby potato cultivar, especially the tuber high-set trait. OBJECTIVE 2: Characterize genetic, molecular, physiological, and biochemical factors that control potato key traits, including disease and other stress resistance, yield, and processing and nutritional qualities. Sub-objective 2A: Delineate mechanisms that mediate small molecules involved in tuber nutritional value and appearance. Sub-objective 2B: Examine the effect of heat-stress on tuber internal defects, phenylpropanoids, and glycoalkaloid metabolism. OBJECTIVE 3: Develop improved molecular diagnostic tools for pathogen detection to facilitate epidemiological studies of important pathogens of potato. Sub-objective 3.A: Develop new tools for rapid identification of Lso and BLTVA phytoplasma in planta and explore the role of new genetic variants of Lso in potato in the Northwest. Sub-objective 3B: Generate and maintain a PMTV-infected Spongospora subterranea population in the greenhouse for use in germplasm screens.

Approach
OBJECTIVE 1: We will use modern breeding methods, and develop and deploy high-throughput phenotyping methods. Drones will collect weekly multispectral images of field trials. Tubers will be phenotyped using digital imaging and a self-built conveyor belt system with sensors to automate phenotyping of tuber number, size, shape, color, eyes, and physiological defects. Parental lines containing disease resistance alleles will be used to develop mapping populations. Populations will be genotyped using DArTseq. Diploids and germplasm from wild potato showing disease resistance will be used to produce self-compatible diploid clones. A factorial breeding population will be used to assess trait correlation and mapping. Joint linkage or association mapping will be used to map QTL and calculate GEBVs for key traits. A major breeding effort will be russet potatoes, but baby and specialty potatoes will be bred with traits including appearance, taste, high tuber number and nutritional value. OBJECTIVE 2: The factors that influence tuber nutritional value and quality, including phenylpropanoids and glycoalkaloids will be analyzed. Time-course studies will use tubers exposed to continuous light. Flavonols will be extracted and measured with LCMS. Gene expression and transcriptomic studies will be conducted if samples show large flavonol increases. Tuber flavonol synthesis will be reprogramed by silencing anthocyanin biosynthesis to test whether this increases flavonols. Terpenoid metabolism will be analyzed in tubers exposed to light. Chlorophyll and carotenoids will be measured by spectroscopy. Glycoalkaloids will be quantitated by LCMS. Relevant genes will be measured by qRT-PCR and network analysis of gene-metabolite interactions visualized. We will develop a lab assay for defects like blackheart and heat necrosis by exposing tubers to varying temperatures. The effect of high temperatures on glycoalkaloids will be assessed in potatoes grown in WA and TX in a randomized complete block design. OBJECTIVE 3: Molecular tools for BLTVA detection will be optimized and validated. Non-potato psyllids found on sticky traps in the Columbia Basin will be analyzed for Lso and transmission to potato tested. At two, four, and six weeks post-inoculation, symptoms will be recorded, and plant tissue will be collected and tested for the presence of Lso to assess whether successful inoculation occurred. To develop and maintain a potato mop top virus (PMTV) infected Spongospora subterranea f. sp. Subeterranea (Sss) population, various potential host plants will be inoculated in the greenhouse with Sss-infested soil. To ensure persistence of the PMTV infected Sss, we will try different methods to ensure inoculum is maintained. One method will cycle potato plants and tomato/N. benthamiana to ensure that the soil always has a potato plant present to maintain PMTV-infected Sss when the tomato needs to be replaced. A second method does not rely on the continual cycling of potato but will grind up the tomato. A third method utilizes PMTV-infected potato obtained each year by planting tubers alongside the tomato or plants to enable transmission to the host plant.

Progress Report
This report documents FY 2024 progress for project 2092-21220-003-000D, “Potato Germplasm Development for Improved Sustainability, Disease Resistance, Nutrition, and Quality”, which began in March 2023. ARS scientists in Wapato, Washington, made progress on all project sub-objectives. All work related to these sub-objectives was completed at the ARS worksite in Prosser, Washington. In support of Sub-objective 1A, ARS scientists utilized a novel, low-cost machine vision workflow to quantify tuber number, size, shape, color characteristics, starch content, and defect frequency of greater than 1,700 field-grown breeding samples. In addition, greater than 30 flights were performed at weekly intervals to capture multispectral drone data from breeding experiment trials in Hermiston, Oregon, Pasco, Washington, and Othello, Washington. Plant height, canopy volume, and multispectral reflectance values were extracted from georectified orthomosaics from all flights. In support of Sub-objective 1B, ARS scientists successfully completed a potato crossing block in the spring of 2024 that generated thousands of recombinant seeds that will be evaluated in future field trials and marker assisted selection. Progress was made by introgressing extreme resistance to Potato Virus Y (PVY), Tobacco rattle virus (TRV), Columbia root knot nematode (CRKN), and Potato cyst nematodes (PCN) into susceptible genetic backgrounds in the processing and fresh market classes. Continuing work will generate multi-parent populations segregating for PVY, TRV, and CRKN resistance in both russet and specialty germplasm. Under Sub-objective 1C, ARS scientists performed a screen of hundreds of diploid accessions (10 individual plants/accession) derived from greater than 15 different potato wild relative species. Varying levels of Potato mop-top virus (PMTV) infection was found in seventeen of these accessions, whereas three accessions derived from S. acuale, S. boliviense, S. chachoense, and S. vernei exhibited no viral infection after 12 weeks of exposure to infected soil. For Sub-objective 1D, ARS scientists grew out and evaluated multi-parent breeding populations focused on transferring PVY and Golden cyst nematode resistance into specialty potato germplasm. Five cultivars of yellow, red, and purple potatoes were hybridized with disease resistant parent ‘Barbara’ and individual clones from each family (100 each) were grown as five-hill plots in Pasco, Washington. Plots were harvested and evaluated for yield, specific gravity, tuber size, shape, color and defects. This population is currently being genotyped. In the spring of 2024, a crossing block was completed that focused on trying to develop specialty lines with a high-tuber set and additional characteristics desired by the baby potato market and consumers, including shape, various skin and flesh colors, and uniformity. Berries from these crosses have been harvested and are being processed to collect true seed that will be planted in greenhouses in Hermiston, Oregon, by collaborators from Oregon State University in order to provide seed for single-hill field trials next year in Klamath Falls, Oregon. Six-thousand seedlings from the 2023 crossing block were planted in Klamath Falls, Oregon this year and will be evaluated this fall. To address Sub-objective 2A, ARS researchers are making transgenic plants with altered anthocyanin metabolism by silencing a gene necessary for the formation of anthocyanins in potatoes. This is providing information about potential ways to increase the amounts of antioxidant and anti-inflammatory compounds in potatoes. Also, in support of Sub-objective 2A, various tubers were treated with light to assess the effect on nutritional value, especially the effect on glycoalkaloids and greening. This included using mass spectroscopy to analyze hundreds of primitive tubers identified as resistant to greening in response to light by ARS scientists in Aberdeen, Idaho. Progress was made toward goals in support of Sub-objective 2B to study the effect of heat on glycoalkaloids in collaboration with researchers from Texas A&M University. Hundreds of potato samples were extracted and then analyzed by liquid chromatography mass spectrometry (LCMS) using a modified method developed this year. The resulting data is now in the process of being analyzed. To evaluate the effect of heat stress on tuber internal physiological defects, tubers were exposed to different temperatures and durations, including lower temperatures and longer exposures to determine if internal heat necrosis can be induced by lab treatment and to assess the overall effect of heat on internal defects. In support of Sub-objective 3A, ARS researchers have been working to generate and maintain alternative psyllid vectors of ‘Candidatus Liberibacter solanacearum.’ Three haplotypes of ‘Ca. L. solanacearum’ have been identified as the causal agent of zebra chip disease in the United States, and two haplotypes are known to be vectored to potato by the potato psyllid. Recently, three new haplotypes of ‘Ca. L. solanacearum’ were discovered in four different non-potato psyllid species by ARS scientists. It is not known whether these newly discovered ‘Ca. L. solanacearum’ haplotypes infect potato or other crops. Only one alternative vector species, Aphalara loca, was collected with the ‘Ca. L. solanacearum’ pathogen in a 2023 survey. In attempts to generate an infected colony of this species, ARS scientists focused on rearing the psyllid on Polygonum aviculare, a newly recognized host plant of A. loca. Additionally, P. erectum seed was obtained from collaborators in the southwest United States and attempts to use this plant to maintain infectious A. loca cultures is underway. While waiting to generate infected colonies, natural populations of the A. loca psyllid are being used to assess transmission of the pathogen to weedy host plants of other Aphalara species, as well as to assess transmission to potato and carrot. This work will determine whether A. loca psyllids are a threat to the potato and carrot industries of the Northwest United States as a vector of ‘Ca. L. solanacearum’. Recent efforts have also focused on identifying infectious Heterotrioza chenopodii, another psyllid vector a newly discovered ‘Ca. L. solanacearum’ haplotype. Work is currently underway to establish this species in culture. Non-infectious cultures of two other Aphalara species are currently maintained for use in controlled transmission trials in subsequent years. To address Sub-objective 3B, ARS researchers generated cultures of PMTV-infected Spongospora subterranea maintained on tomato plants. Currently, there are no commercial potato cultivars resistant to PMTV. Greenhouse screens are currently underway to identify PMTV-resistant material. However, a steady inoculum source is needed to ensure long-term success of these greenhouse screening assays. Greenhouse cultures of S. subterranea, grown on tomato, are used to generate this continual inoculum source. Initial maintenance of our cultures through cycling tomato plants led to depletion of S. subterranea populations, suggesting that the protist was staying inside tomato root tissues. To avoid this, tomato plants are now cycled by overlapping time, so that as one plant gets old, a young tomato is transplanted into the pot to help increase and generate the S. subterranea population. Success of this method will be confirmed in upcoming months. To find a better host plant for maintaining the S. subterranea cultures, various grass species were screened for susceptibility to S. subterranea and Potato mop-top virus. Unfortunately, no species were identified, indicating that Solanaceae are the best host plants for maintaining a culture of this pathogen complex.

Accomplishments
1. Procedure to assess the glycoalkaloid spiking potential of new potato breeding lines. Certain environmental stimuli such as light can cause potato tuber greening and toxic spikes in potato glycoalkaloids (GLKs), leading to health concerns, culling and trade issues. A major concern for the industry is the potential for GLK amounts to unpredictably spike in response to currently unrecognized environment stimuli, especially in new cultivars grown on a much wider scale following commercialization. ARS researchers in Wapato, Washington, at the worksite in Prosser, Washington, in collaboration with Washington State University scientists, evaluated GLK metabolism and greening in response to cold or light in transgenic lines and in 20 cultivars. They found that greening can occur without any increase in GLKs and that even after a week of exposing tubers to light, GLK amounts remained acceptable in many cultivars. White potatoes with more active isoprenoid metabolism had larger increases in GLKs following light or cold treatment compared with white potato cultivars with less active isoprenoid metabolism or yellow-flesh potatoes with active isoprenoid metabolism. These findings suggest that to decrease the likelihood of unpredictable GLK spikes, potato breeding programs can assess the spiking potential of new breeding lines by measuring the combined increase in GLKs, carotenoids, and chlorophyll following light or cold treatment.

2. Deployment of a novel, low-cost data collection workflow to phenotype potato breeding lines. Incorporation of novel disease resistance traits with other quality traits is a major challenge in tetraploid breeding systems like potato. Traditional potato breeding methods rely on slow, labor-intensive methods to measure traits which cannot be applied until the third or fourth year of the breeding program. ARS researchers in Wapato, Washington, at the worksite in Prosser, Washington, developed low-cost, high-throughput workflow to measure and phenotype potato tubers for critical traits including yield, shape, skin characteristics, starch content, and susceptibility to defect. Using this workflow, they found that both maternal and paternal parents influence yield and tuber shape, but only the paternal line influences nematode resistance. These results provide useful information on trait inheritance, while the validation of this high-throughput workflow will decrease the amount of time needed to produce high-quality potato breeding lines by allowing high-throughput screening of critical traits in the first or second field seasons.

3. Development of a high-throughput assay to detect three plant pathogens in the beet leafhopper vector. Beet leafhopper is a vector of three plant pathogens – Phytoplasma trifolii, beat curly top virus, and Spiroplasma citri – that cause economic damage to vegetable crops. ARS researchers and Washington State University extension personnel monitor populations of beet leafhopper throughout the season to support areawide management of this vector, but previous methods for pathogen detection were too time-consuming and expensive to allow up-to-date testing of all three pathogens within vector populations. ARS researchers in Wapato, Washington, at the worksite in Prosser, Washington, in collaboration with Washington State University scientists, developed and validated a high-throughput and cost-effective method to test beet leafhopper for all three plant pathogens. They used this method to provide Washington State University Extension with weekly data on prevalence of pathogens within beet leafhoppers collected from various vegetable growing regions of the state. These weekly updates were provided to growers through the Washington State University Decision Aid System online tool to allow information on prevalence of plant pathogens to be incorporated into seasonal insect pest management programs.

Review Publications
Foutz, J.J., Cooper, W.R., Swisher Grimm, K.D., Crowder, D. 2024. Seasonal and lifecycle changes in behavior affect the trapping efficiency of an insect vector, Circulifer tenellus (Hemiptera: Cicadellidae). Annals of the Entomological Society of America. 117(3):199-205. https://doi.org/10.1093/aesa/saae011.
Goraya, M., Yan, G., Whitworth, J.L., Swisher Grimm, K.D. 2023. Advancing nematode identification on potato: An isothermal recombinase polymerase amplification assay for stubby root nematode, Paratrichodorus allius. American Journal of Potato Research. 101:52-64. https://doi.org/10.1007/s12230-023-09940-4.
Tuggle, C.K., Clarke, J.L., Murdoch, B.M., Lyons, E., Scott, N.M., Mckay, S., Lipka, A., Fulton, J., Hess, A., Lubberstedt, T., Fragomeni, B., Rowan, T., Mccarthy, F., Guadagno, C., Goddard, E., Das Choudhury, S., Sheehan, M., Kramer, L., Feldman, M.J., Daigle, C., Steibel, J.P., Benes, B., Murray, S., Riggs, P., Thompson, A., Hagen, D., Thornton-Kurth, K., Van Tassell, C.P., Campbell, J.D., Dorea, J., Chung, H., Dekkers, J.C., Ertl, D., Lawrence-Dill, C.A., Schnable, P.S. 2024. Current challenges and future of agricultural genomes to phenomes in the USA. Genome Biology. 25:8. https://doi.org/10.1186/s13059-023-03155-w.
Feldman, M.J., Park, J.B., Miller, N., Wakholi, C., Greene, K.B., Abbasi, A., Rippner, D.A., Navarre, D.A., Schmitz Carley, C., Shannon, L.M., Novy, R.G. 2024. A scalable, low-cost phenotyping strategy to assess tuber size, shape, and the colorimetric features of tuber skin and flesh in potato breeding populations. The Plant Phenome Journal. 7(1). Article e20099. https://doi.org/10.1002/ppj2.20099.
Lachowiec, J., Feldman, M.J., Matias, F.I., LeBauer, D., Gregory, A. 2024. Adoption of unoccupied aerial systems in agricultural research. The Plant Phenome Journal. 7(1). Article e20098. https://doi.org/10.1002/ppj2.20098.
Zhu, M., Bibi, S., Sun, Q., He, Y., Xue, Y., Su, Y., Navarre, D.A., Du, M. 2024. Dietary purple potatoes shape gut microbiota and ameliorate colitis in IL-10-deficient mice. Journal of Functional Foods. 119. Article 106321. https://doi.org/10.1016/j.jff.2024.106321.
Swisher Grimm, K.D., Gorman, C., Crosslin, J. 2023. New assays for rapid detection of beet leafhopper-associated plant pathogens, 'Candidatus Phytoplasma trifolii', Beet curly top virus and Spiroplasma citri. Plant Disease. 107(12):3958-3966. https://doi.org/10.1094/PDIS-04-23-0769-RE.
Gorman, C., Crowder, D.W., Swisher Grimm, K.D. 2023. A high-throughput plate method for nucleic acid extraction from beet leafhopper (Hemiptera: Cicadellidae) and potato psyllid (Hemiptera: Triozidae) for pathogen detection. Journal of Economic Entomology. 116(5):1876-1884. https://doi.org/10.1093/jee/toad153.