Location: Forage Seed and Cereal Research2017 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:
New molecular markers were identified and developed for resistance to powdery mildew and downy mildew in hop (Objective 1). New genetic maps, containing in excess of 2000+ genetic markers, for two mapping populations were developed. Fifteen (15) putative molecular markers linked to powdery mildew resistance were identified; all significantly linked markers were found within 3 closely linked quantitative trait loci (QTLs) on the same chromosome. The putative genes located nearby (within 1 centimorgan) were also identified. Markers associated with resistance to downy mildew were located upon three different linkage groups—lending support to observations that resistance to downy mildew is under quantitative genetic control. Previously identified molecular markers linked to expression of resistance to downy mildew were double validated using a second set of 150 hop varieties and found to be consistent with identifying offspring or parents possessing resistance. Molecular markers linked to short-stature hop growth patterns—so called "dwarf hops" were identified in 2017. Phenotypic data collection along with development of molecular markers, genetic mapping and QTL studies identified regions of the hop genome controlling the expression of “dwarfing” in hop. One chromosome contained all the major regions determining whether an offspring was short versus tall as well as the regions determining how short the dwarf plant grew. These chromosomal regions were investigated at the genomic level to see what proteins were present and if these proteins were potentially responsible for the dwarfing trait. Several proteins were identified with one protein standing out as being responsible for dwarfism in other plant species. Additional genomic work on traits of economic importance in hop was also completed this year. Previously reported completion of an early draft hop genome enabled the identification of genomic regions responsible for expression of sex in this dioecious crop. In addition, approximately 25,000 genes and their location on DNA scaffolds in the USDA hop cultivar ‘Teamaker’ (the draft version of this genome of which was published online at http://hopbase.cgrb.oregonstate.edu/) were annotated and made public. Identification of Y-specific chromosome regions as well as regions of the X and Y chromosomes that pair and recombine were identified. Proteins expressed from genes located on these regions were identified and the expression of these genes across male and female plants housed in the USDA-ARS hop germplasm collection determined. Some genes showed dramatic differences in expression between sexes. In addition, a large set of male-specific molecular markers were identified that will enable simple molecular assays used to screen out male offspring from breeding nurseries. New genomic sequencing research was initiated and completed that utilized new third generation sequencing platforms. Previous work on sequencing and assembling the hop genome illustrated the difficulties of genomic work on species with large portions of the genome consisting of repetitive DNA and high heterozygosity. DNA heterozygosity arises when parents are genetically diverse, such as is the case in hop, which has separate male and female plants. An ARS-Oregon State University-industry partnership led to the development and assembly of a far superior draft genome of the widely used USDA-ARS hop variety ‘Cascade’. The previously developed draft genome of hop covered approximately 63% of the hop genome, leaving large portions undiscovered. This new study was designed to sequence through all repetitive regions of the genome and allow for the sequencing of both strands of DNA making up a chromosome; the latter capability enabling the sequencing and assembly of heterozygous regions. One of the primary goals of this project is the development of disease resistant hop varieties (Objective 2). Hop variety development may take up to 15 years before public release due to the perennial nature of the crop. The first stage of selection for disease resistant hop cultivars with crosses made in 2012 was completed in 2017. Fourteen genotypes from 10 different families were selected from small plot nurseries for advancement to large-scale plots for continued observations on yield, agronomics and brewing potential. These genotypes resulted from selection performed under greenhouse conditions, short-trellis ‘pot-in-pot” nurseries and single hill plots for disease resistance, yield potential, growth habits and cone aroma and shape. They will undergo observations in large plots for 3-4 more years at which point one or several will be replanted into 2-3 acre sized plots for commercial observations, chemistry and pilot brewing. Lines that prove agronomically successful and show brewers acceptance will be released to the public. The final year of work was completed to clarify the ecology of overwintering of the powdery mildew fungus (Objective 3, sub-objective 3A). To date, the research shows that the pathogen survives from year-to-year at only a low level (less than 5% of fields) in the western U.S. The research also identified the time of the season where infection by the pathogen is most likely to lead to its survival to the next year and mediating factors that influence the likelihood of pathogen survival. Data collection was completed from commercial fields and statistical analyses were largely completed to further identify and quantify risk factors for perennation of the powdery mildew fungus. Factors that were discovered to influence the degree of pathogen survival from year-to-year in a given field included the severity of disease in the previous season, prior occurrence of overwintering in the same or adjacent field, winter temperature, and certain grower cultural practices. A collaboration with a statistician at Oregon State University was continued to further develop a mathematical model to predict how disease spreads from these overwintering locations amongst fields. The present model predicts very well how disease develops at the landscape level and which fields are most important for spreading disease. Thus, the research provides guidance on where the fungus is most likely to persist over the winter and how this survival is likely to affect disease development in both nearby and distant fields. This information is critical for understanding and deploying control measures appropriately. A new discovery was made that hop seed may be infested with the sexual stage of the powdery mildew fungus. Extant quarantine regulations are in place in the Pacific Northwestern U.S. to prevent introduction of new strains of the pathogen and this life stage specifically. However, these regulations explicitly exempt seed. This research indicates that current quarantine regulations may need to be reevaluated to mitigate the risk of new introductions of the pathogen via this route. Studies were completed that describe the mating system of the hop downy mildew pathogen (Objective 4, sub-objective 4A). The pathogen was found to produce a hardy, long-lasting survival structure when only one strain of the organism was present, not two strains as often is found in related pathogens. This has important implications for understanding how the pathogen may survive from year to year and breeding strategies for developing resistant varieties. Related experiments were planned and successfully executed to extract DNA and characterize the genetic diversity of a large collection of samples of the downy mildew pathogen according to the project plan. A collaboration with other ARS researchers was established to assist with data analysis, which is nearly complete. Manuscript preparation is underway. The final phases of research to characterize the genetic diversity and relatedness among populations of the powdery mildew fungus was launched. Sequencing of RNA copies of genes was nearly completed to identify genetic variants in a worldwide collection of the fungus. Preliminary analyses were begun, which indicate that there is variation between populations of the pathogen in different geographic regions of the world but limited variation within certain regions. This may influence how researchers approach screening of plant materials for disease resistance and for inferring migration of the pathogen. Additional research beyond the original scope of the project plan also was launched. Following a disease control failure following application of one of the most widely used downy mildew fungicides in the Pacific Northwest, a new project was begun to determine if resistance to phosphorous acid exists within the hop downy mildew pathogen. Preliminary studies have detected insensitivity to greater than twice the allowable field rate of the fungicide and cross resistance with another fungicide, fosetyl-Al. These early results have been communicated to industry. Further, new research was begun to quantify the impact of various grower production practices (fertility rates, irrigation practices, and post-harvest practices) on levels of powdery mildew, downy mildew, a complex of arthropod pest, and crop quality factors. This research is expected to provide data to support development of best management practices for priority disease and pest issues affecting stakeholders.
1. Seedborne dissemination of novel strains of the hop powdery mildew pathogen. Powdery mildew is the most costly disease affecting the U.S. hop industry, with disease-related costs exceeding 15% of crop value annually. Quarantine measures are in place in the Pacific Northwestern U.S., the primary production region of the crop, to prevent the introduction of novel strains of the pathogen but these quarantine measures explicitly exempt import of seed. ARS researchers in Corvallis, Oregon, with collaborators at the University of Minnesota, discovered that hop seed can be contaminated with the powdery mildew fungus. Strains of the pathogen that are not established in the western U.S. were identified on seed lots shipped into the region. This research indicated that extant quarantine rules may need to be reevaluated to mitigate the risk of new introductions of the pathogen.
2. Rapid response to emergence of virulent strains of the hop powdery mildew fungus. The most efficient and cost effective means of controlling plant diseases is through planting of resistant varieties that do not develop disease. The disease powdery mildew of hop was controlled for 15 years on a substantial portion of the U.S. industry in this manner until new strains of the pathogen emerged that could overcome certain resistant varieties. ARS researchers at Corvallis, Oregon, with collaborators at Oregon and Washington State Universities, identified the distribution of these new strains of the pathogen in the region, clarified the susceptibility and appropriate level of management intervention needed on formerly resistant varieties, and discovered extant varieties and breeding lines that still remain resistant. This research provided the biological basis for producers to understand and appropriately respond to this new disease threat in the near term. It has also provided the foundation for long-term mitigation of the problem by accelerating breeding efforts to develop new, resistant varieties.
3. Development and assembly of a complete draft genome of hop. Identification of molecular markers for use in selection regimes is highly dependent upon the use of a complete draft genome covering all chromosomal regions. Previous quantitative trait locus (QTL) studies identifying molecular markers in hop utilized a draft assembly covering only 63% of the genome—leaving open the possibility that the best molecular markers would be overlooked. ARS scientists in Corvallis, Oregon along with collaborators at Oregon State University and Pacific Biosciences, sequenced and assembled a new draft hop genome that covers approximately 98% of the genome. This research discovered that up to 3% of the hop genome consists of structural variants of DNA sequences and also identified highly repetitive DNA consisting of transposable elements and other repeat structures. Previous draft genomes were made up of relatively short stretches of DNA sequence data with average assembled size of such sequences being 40,000 base pairs while the new draft genome has an average size of assembled sequences equaling 750,000 base pairs. This new draft genome will enable the precise identification of molecular markers and genes linked to traits of economic importance in hop as well as enable other molecular tools such as unknown variety identification.
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Ojiambo, P.S., Gent, D.H., Mehra, L.K., Christie, D., Magarey, R. 2017. Focus expansion and stability of the spread parameter estimate of the power law model for dispersal gradients. PeerJ. doi: 10.7717/peerj.3465.