Location: Forage Seed and Cereal Research2018 Annual Report
1a. Objectives (from AD-416):
Objective 1: Identify new genetic markers for selecting resistant hop germplasm to downy and powdery mildews. Objective 2: Develop new disease resistant germplasm for public release. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Sub-objective 3.A: Describe the ontology of crown bud development, susceptibility of crown buds to powdery mildew in different developmental stages, and dynamics of flag shoot emergence. Sub-objective 3.B: Develop a PCR assay to rapidly identify mating type in P. macularis; determine prevalence of mating types among isolates of P. macularis in the Pacific Northwest. Objective 4: Develop and apply genotyping approaches to assess the diversity, geneticdifferentiation, and sexual recombination in the downy mildew and powdery mildew pathogens. Sub-objective 4.A: Identify and develop simple sequence repeat markers in Pseudoperonospora humuli and elucidate the degree of diversity, selfing, and population differentiation within and among population at multiple hierarchical scales. Sub-objective 4.B: Identify polymorphic loci among isolates of Podosphaera macularis and characterize the genetic diversity, population structure, and relatedness of the population in the Pacific Northwest U.S. to other populations of the pathogen in the world.
1b. Approach (from AD-416):
Objective 1: Identify molecular markers associated with plant resistance to P. humuli and P. macularis. Progeny developed from crosses of resistant and susceptible parents will be screened by pathogen challenge, and single nucleotide polymorphic (SNP) marker identification and genotyping-by-sequencing will be performed. Objective 2: Development of multiple pathogen resistant germplasm or varieties. Progeny of crosses of parental material that possess relative resistance to powdery and downy mildews will be successively challenged with each disease to identify germplasm with enhanced resistance to both. Resistant germplasm that possess excellent agronomic and brewing characteristics will be released. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Hypothesis 3.A: Successful perennation of the powdery mildew fungus occurs via infection of juvenile crown buds and such crown buds develop asynchronously. Management factors that reduce late season severity of powdery mildew will reduce overwintering survival of the pathogen. Crown bud development phenology will be determined and plants will be challenged with powdery mildew at selected stages. Treatment at different stages will be evaluated. Hypothesis 3.B: The absence of the ascigerious stage of Podosphaera macularis in the Pacific Northwestern U.S. is due to the absence of one of requisite mating types of the fungus. PCR amplification of conserved regions in MAT1-1 and MAT1-2 loci of P. macularis will be optimized and pathogen isolates collected from a variety of cultivars and hop yards in the Pacific Northwest will be tested to determine the frequency of each mating type. Objective 4: Develop and apply genotyping approaches. Hypothesis 4.A: P. humuli is heterothallic, possessing a high degree of genetic diversity in the Pacific Northwestern U.S, and the population is structured at the scale of individual fields. P. humuli isolates will be obtained from three hop yards in western Oregon. After suitable SSR loci and primers are developed from pyrosequences, genotyping will be performed by capillary sequencing of 7 to 10 SSR loci per isolate. Hypothesis 4.B: The population of Podosphaera macularis in the Pacific Northwestern U.S. exhibits a low degree of genetic diversity or structure based on geography or cultivar host. Multiple nuclear loci will be identified, PCR-amplified, and sequenced from Pacific Northwest, northeastern U.S., and European isolates. Population genetic parameters will be calculated and differentiation among geographic populations will be estimated.
3. Progress Report:
This is the final report for project 2072-21000-0046-00D, which expired March 2018 and was replaced by project 2072-21000-051-00D, "Breeding, Genomics, and Integrated Pest Management to Enhance Sustainability of U.S. Hop Production and Competiveness in Global Markets." For additional information, see the new project report. Under Objective 1, significant progress was made in identifying markers linked to resistance to downy and powdery mildew in hop. Bi-parental crosses were made for evaluation of downy mildew resistance, and offspring cloned for greenhouse and field-based disease resistance experiments in Oregon and Washington. Offspring and parents were genotyped-by-sequencing. The resulting genotype and resistance data were used in quantitative trait loci (QTL) studies to identify genome regions containing resistance genes. Markers linked to downy mildew resistance were observed on three different linkage groups confirming previous research suggesting multi-genic quantitative control. Markers located near QTLs were identified and reported in a peer-reviewed manuscript. Additional research with the same plant material was coupled with disease resistance data on a collection of world hop cultivars and new markers identified and reported in a peer-reviewed manuscript. Objective 1 also included studies on plant resistance to powdery mildew (PM) in hop. Bi-parental crosses were made between PM-resistant and a PM-susceptible USDA germplasm. The offspring and parents from this cross were evaluated for resistance under controlled conditions in the greenhouse in replicated experiments. All plants were inoculated with controlled levels of spores and plant response taken two weeks after inoculation. Genotyping-by-sequencing was performed on all plants and the resulting molecular markers utilized to develop genetic maps. Quantitative trait loci studies were performed and identified one major QTL with several minor QTLs located on the same chromosome. Several putative genes were found within QTLs and are known to be active in plant disease response in other species. These genes are therefore logical targets for development of molecular markers useful for marker assisted selection, which may accelerate cultivar development. ARS efforts to develop disease resistant hop germplasm (Objective 2) made significant steps towards this goal. Hop breeding is a long-term process with cultivars taking up to 20 years to develop and release. The objectives of this project involved making crosses with resistance to both powdery and downy mildews and subsequent steps at selecting lines that exhibit resistance as well as high yields and good brewing characteristics. Thirteen experimental lines were selected from these crosses and subsequently advanced into larger plots in three states. First year observations on selected lines show excellent field resistance to both pathogens, and several of these individuals show promise as future hop cultivars. Public release of these putative cultivars requires additional observations in larger plots and multiple environments. Research from Objective 1 was extended to discern the genetic control of adaptation to short-trellis production systems. The results of this study showed two major QTLs controlling the trait, confirming the results from previous research. Advances in hop genomics enabled the location of linked markers on genome scaffolds along with candidate genes that may control the trait. The results for this study were published in peer reviewed manuscripts. Under Sub-objective 3A, significant progress was made in identifying factors that enable the powdery mildew fungus to persist from year-to-year, which formed the basis of improved management of the disease industry-wide. Progress was also made in clarifying why the sexual stage of the pathogen is absent in the western U.S. but present elsewhere in the world where the disease occurs. Under Sub-objective 3B, molecular assays were developed that can be used to rapidly identify what is known as mating types of the pathogen (analogous to gender in animals). Using these assays, ARS researchers demonstrated that only one of the two mating types of the fungus required for sexual mating is present in the western U.S. This information served as the scientific basis for stakeholders to work with the Departments of Agriculture in Washington, Idaho, and Oregon to revise and harmonize quarantine rules in the region to prevent introduction of the second mating type into the region. Under Sub-objectives 4A and 4B, significant progress was made in describing the genetic structure of populations of the powdery mildew and downy mildew pathogens, and the implications of this for disease management in both the near and long-term. The research demonstrated that the downy mildew organism is self-fertile and the pathogen lifecycle is dominated by clonal reproduction. ARS researchers also discovered that the genetic fingerprints of the pathogen in commercial fields is related to the source of the original planting stock. This has important implications for how producers source planting material and the risk of spread of novel strains of the organism long-distance in planting stock. Similarly, for the powdery mildew organism, researchers discovered that three races of the pathogen are predominate in the U.S., and identified resistance genes in publically available material that can provide resistance to these races. They discovered that the likely origin of the pathogen in the western U.S. was originally from Europe. However, in the U.S., ARS researchers also found evidence that populations of the organism are distinct on feral versus cultivated plants, and that spread of the organism on planting stock is probable. These findings have informed and accelerated breeding efforts for the disease, as well as informed disease management efforts related to where and how planting stock is produced. Stakeholders now have new germplasm, candidate molecular markers linked to resistance to the two most important diseases of hop, new knowledge that has modified quarantine rules, and enhanced management systems to improve management of the two most important diseases limiting hop production efficiency, sustainability, and profitability.
1. Population genetic structure and spread of the hop downy mildew organism. Downy mildew is one of the most devastating diseases of hop in wet, humid production regions worldwide. Little is known about the genetic diversity and structure of the pathogen population, which limits abilities to appropriately breed for improved disease resistance and mitigate the disease. ARS scientists in Corvallis, Oregon, conducted the most complete analysis of the pathogen genetic diversity to-date, and found that there is limited diversity in the pathogen's population in the main production regions in the western U.S. However, evidence of persistence over time of the same population associated with planting stock was discovered. This research informs current and future breeding efforts for the disease, but in the immediate term, the research also points to infected planting material as an important means of spread and persistence of the pathogen that must be mitigated.
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