1a. Objectives (from AD-416):
Objective 1: Apply genetic analyses, metabolic engineering, and targeted metabolic profiling to elucidate genetic, molecular, and biochemical factors governing host disease resistance and accumulation of select phytonutrients and vitamins in potatoes. Sub-objective 1.A. Characterize molecular and biochemical factors that modulate phytonutrient content. Sub-objective 1.B. Characterize molecular and biochemical mechanisms involved in disease/pest resistance. Objective 2: Evaluate, breed, and release potato germplasm with increased amounts of phytonutrients, which are suitable for the processing and fresh potato market, as well as for niche markets. Objective 3: Identify and release germplasm or varieties with improved resistance to powdery scab, black dot, Columbia root-knot nematode, zebra chip, potato mop top virus, potato cyst nematode, and examine the role of micronutrients in host resistance to Verticillium wilt. Sub-objective 3.A: Nematodes: Focus on identifying and developing germplasm, including trap crops, that can provide superior control options for Columbia root-knot nematode or Potato Cyst Nematode. Sub-objective 3B: Soil borne pathogens: Develop superior germplasm or management options for soil borne pathogens including powdery scab, potato mop top virus, black dot and Verticillium wilt. Objective 4: Determine available host-plant resistance and epidemiological parameters, and develop diagnostic tests for emerging pests and pathogens of potato such as zebra chip.
1b. Approach (from AD-416):
Objective 1: We will utilize molecular physiology approaches, including measuring gene expression, enzyme activity and metabolite pools by hyphenated techniques. Structural genes and regulatory genes will be assessed using transient assays or stable transgenics. The phenylpropanoid pathway will be a focus. HQT expression will be reduced using RNAi. LCMS will be used to assess differences in phenylpropanoids between wild type and silenced lines and the expression of at least 10-20 phenylpropanoid genes measured by qPCR. Another gene targeted for silencing will be dihydroflavonol-4-reductase (DFR). LCMS and GCMS analysis will be used to examine how phenylpropanoid and primary metabolism is reprogrammed in plants with altered DFR metabolism. MYB transcription factors will be identified in silico based on phylogenetic and protein similarity with known transcription factors. Function will be assessed in transient and stable assays. Compounds that cause the hatching of potato cyst nematode eggs will be partially purified from root extracts using chromatographic methods. Objective 2: Tuberling populations will be assembled and grown two successive seasons in the Klamath Basin of Oregon in unreplicated plots. Promising material will be analyzed for carotenoids, anthocyanins, antioxidants, and a range of other metabolites to select clones with high phytonutrient content. Statistically the data will be analyzed as a mixed model with locations, clones and interaction as fixed effects and reps within locations as random effects. We will use molecular markers to characterize hybrids and assure that we intercross only duplex Zep1 hybrids. Objective 3: We will combine PVY extreme resistance and CRKN resistant germplasm. The genetic nature will be explored by determining segregation ratios in reciprocal crosses. Mitochondrial fingerprinting will be expulsed as a diagnostic genetic marker of the restored phenotype. Crosses will be made to select a less spiny version of Solanum sisymbriifolium for use as a PCN trap crop. A. rhizogenes will be used to attempt to make a version of the plant with greater root mass. Hatching assays will be used to screen for other plants that may be a superior PCN trap crop. Crosses will be made to generate potatoes with resistance to Black dot and Powdery scab and evaluated in field trials with a randomized complete block design with four replications and ten plants per replication. The crown and root will be scored for degree of galling and sclerotia. The effect of micronutrient supplements on Verticillium wilt resistance will be assessed in field and greenhouse trials. Macro and micronutrients will be applied in-furrow. Objective 4: Psyllids collected during the survey and additional insects collected in the Pacific Northwest will be subjected to high resolution melt (HRM) analysis of the cytochrome oxidase gene in order to differentiate genetic variants of the psyllid. Extracts will be tested by PCR methods reported in the literature at dilutions up to 1,000 to determine level of sensitivity and reliability of the various methods on different host plant tissues.
3. Progress Report:
This new project replaces 5354-21220-010-00D, "Potato Germplasm Enhancement Through Trait Discovery, Genetic Evaluation and Incorporation", and has just been initiated. Progress is underway. As described in our final report for the previous project, much of the new effort builds upon our previous discoveries and progress. Additional gene expression and metabolite relationships during development have been quantitated in pathways not previously analyzed. These results reveal additional phytonutrients are expressed at higher levels early in tuber development. Efforts are underway using transient assays and gene expression analysis to determine which transcription factors are involved in the regulation of tuber antioxidants. Screening is underway to search for a faster growing form of the PCN trap crop S. sisymbriifolium. A faster growing form could potentially allow growers to plant a second non-trap crop in the same season to generate an economic return. Evaluation of potato psyllid populations from around the country is underway to better understand aspects of the zebra chip disease, including severity and overwintering mechanisms. Mineral studies have been initiated to evaluate the effect on potato soil-borne pathogens.