Progress 03/10/04 to 06/15/08
Outputs
Progress Report Objectives (from AD-416) Identify, sequence, map and characterize hop genes conferring specific phenotypic functions of economic importance. Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection. Develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. Characterize molecular and genetic factors that impact the host/pathogen interactions in contrasting Pacific Northwest hop production environments. Characterize and quantify environmental and cultural conditions that impact the occurrence and spread of hop pathogens, particularly hop powdery and downy mildews. Integrate the results of this research with that of cooperating hop researchers to develop a coordinated production strategy that reduces production input costs. Approach (from AD-416) Use conventional and molecular breeding techniques, PCR methodologies, GC/MS spectrophotometric, and HPLC techniques. Study the genetics of disease resistance and other traits of economic importance. Identify genetic sources of disease resistance and superior agronomic traits from current and new germplasm. Grow and harvest hops commercially, obtain disease resistance ratings, yield, quality, brewing, and taste panel evaluation. Identify genetically diverse parents for heterosis. Release germplasm for public use. Previous Projects: 5358-21000-019-00D (Exp 4/01) . Replacement for 5358-21000-030-00D (Exp 03/04). FY04 Program Increase $201,306. Add 1 SY. Replaced by 5358-21000-040-00D (6/08) normal progression. Significant Activities that Support Special Target Populations This project was terminated 6/15/08 (Normal Progression) and replaced by CRIS project 5358-21000-040-00D approved through the OSQR process. A number of crosses that address Objective 1 and 2 were made in Fiscal Year 2008 and nurseries from these crosses were established in the field but clones from the selected individuals were not taken. Molecular markers analyses were performed on two populations designed to map the hop genome for resistance to powdery mildew. We observed two molecular markers tightly associated with the expression of plant resistance to powdery mildew in hop. Nevertheless, insufficient marker coverage of the genome was observed so additional markers are in the process of being examined. An international collaboration was established with other hop geneticists to test new molecular technology using these same populations. In support of Objective 3, bioassay plants were deployed in a hop yard and preliminary infection risk models were developed based on weather and inoculum variables. Efforts to evaluate and validate the model were initiated. A PCR assay specific to the downy mildew pathogen was developed. Field evaluation and validation of the assay are in progress, as well as parallel studies with Sclerotinia sclerotiorum with international collaborators. Data set collection is ongoing in commercial hop yards to identify factors associated with development of powdery mildew on cones; a preliminary cone infection model was developed using historical data sets. Field studies to identify the phenology and population dynamics of arthropod pests and natural enemies in hop yards in relation to the timing of sulfur applications are underway in support of Objective 4. In Year 1 of the regional project, fungicide programs that may exacerbate or suppress spider mite populations were identified. This research contributes to the NP301, Component 2. This research also contributes to NP303, Plant Disease, Component 2 and Component 3.
Impacts
(N/A)
Publications
- Gent, D. H., Nelson, M. E., George, A. E., Grove, G. G., Mahaffee, W. F., Ocamb, C. M., Barbour, J. D., Peetz, A., and Turechek, W. W. 2008. A decade of hop powdery mildew in the pacific northwest. Online. Plant Health Progress doi:10.1094/PHP-2008-0314-01-RV.
- Gent, D.H., Turechek, W., Mahaffee, W.F. 2007. Sequential sampling for estimation and classification of the incidence of hop powdery mildew I: Leaf sampling. Plant Disease. 91:1002-1012.
- Walton, V.M., Dreves, A., Gent, D.H., James, D.G., Martin, R.R., Chambers, U., Skinkis, P.A. 2007. Relationship between rust mites, Calepitrimerus vitis (Acari: Eriophyidae), bud mites Colomeris vitis (Acari: Eriophyidae) and short shoot syndrome in Oregon vineyards. International Journal of Acarology. 33:307-318.
- Gent, D.H., Turechek, W., Mahaffee, W.F. 2007. Sequential sampling for estimation and classification of the incidence of hop powdery mildew II: Cone sampling. Plant Disease. 91:1013-1020.
- Bassil, N.V., Gilmore, B.S., Oliphant, J.M., Hummer, K.E., Henning, J.A. 2007. Genic SSRs for European and North American Hop (Humulua lupulus L.). Genetic Resources and Crop Evolution. Available: www.springerlink. com/content/d000k04454n55v2p/fulltext.pdf
- Gent, D.H., Nelson, M.E., Grove, G.G. 2008. Persistence of Phenylamide Insensitivity in Pseudoperonospora humuli. Plant Disease.92(3):463-468.
- Henning, J.A., Haunold, A., Townsend, M.S., Gent, D.H., Parker, T. 2008. Registration of 'Teamaker' Hop. Journal of Plant Registrations.2(1):13-14.
- Gent, D.H., Turechek, W., Mahaffee, W.F. 2008. Spatial and Temporal Stability of the Estimated Parameters of the Binary Power Law. Phytopathology. 98:1107-1117.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Identify, sequence, map and characterize hop genes conferring specific phenotypic functions of economic importance. Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection. Develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. Characterize molecular and genetic factors that impact the host/pathogen interactions in contrasting Pacific Northwest hop production environments. Characterize and quantify environmental and cultural conditions that impact the occurrence and spread of hop pathogens, particularly hop powdery and downy mildews. Integrate the results of this research with that of cooperating hop researchers to develop a coordinated production strategy that reduces production input costs. Approach (from AD-416) Use conventional and molecular breeding techniques, PCR methodologies, GC/MS spectrophotometric, and HPLC techniques. Study the genetics of disease resistance and other traits of economic importance. Identify genetic sources of disease resistance and superior agronomic traits from current and new germplasm. Grow and harvest hops commercially, obtain disease resistance ratings, yield, quality, brewing, and taste panel evaluation. Identify genetically diverse parents for heterosis. Release germplasm for public use. Previous Projects: 5358-21000-019-00D (Exp 4/01) . Replacement for 5358-21000-030-00D (Exp 03/04). FY04 Program Increase $201,306. Add 1 SY. Accomplishments Association Mapping and Identification of Molecular Markers Linked to Downy Mildew Resistance. The USDA-ARS Forage Seed and Cereal Research Unit identified genetic markers linked to resistance to Hop Downy Mildew. This is significant because traditional germplasm development methods for hop downy mildew resistance have proven insufficient for genetic improvement for this trait. Three years of leaf area incidence of downy mildew infection were collected on 100 hop accessions representing the breadth of diversity in hop. A total of 43 genetic markers that were tested shoed statistical linkage with downy mildew severity and two of the 43 were clearly associated with resistant genotypes. This discovery will greatly aid in efforts to develop new downy mildew resistant hop germplasm. This accomplishment addresses National Program 301 � Plant Genetic Resources, Genomics and Genetic Improvement; Component 2� Crop Informatics, Genomics, and Genetic Analyses, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits. It also address National Program 303�Plant Diseases; Component V�Host Plant Resistance to Disease. Integration of Powdery Mildew Control and Conservation Biological control: The USDA-ARS Forage Seed and Cereal Research Unit in collaboration with Washington State University identified powdery mildew management programs that enhance biological control of spider mite by natural enemies. This is significant because fungicides commonly used for powdery mildew management can increase populations of and damage by spider mites in numerous crops, leading to increased use of pesticides. Fungicide programs were evaluated at multiple field locations, and direct and indirect effects of fungicides on spider mites and natural enemies were documented. This discovery may allow growers to minimize pesticide use by enhancing the reliability of conservation biological control of spider mites, and result in optimized fungicide use, reduced input costs for producers, and enhanced environmental protection. This accomplishment addresses National Program 303 � Plant Diseases; Component I - Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens; Problem Statement 1B: Detection, Identification, Characterization, and Classification of Pathogens. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 23 Number of Newspaper Articles,Presentations for NonScience Audiences: 17
Impacts
(N/A)
Publications
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The most critical problem facing the U.S. hop industry is the need for hop varieties that provide superior brewing quality and that contain resistance to two serious diseases, powdery (PM) and downy mildew (DM). Although DM has been a problem in U.S. hop producing regions for many years, hop PM was first identified in the Pacific Northwest growing regions in 1997. Downy mildew breeding has not progressed significantly over the past half century due to the highly variable nature of disease expression in hops. The increased costs associated with controlling these two diseases threaten the economic viability of the entire U.S. hop industry. Presently, the only way to control PM and DM is by growing resistant varieties or applying expensive fungicides, as many as eight times per season. The
costs of these applications can easily exceed the gross receipts for the crop. When PM first emerged in U.S. hop production fields, only a few resistant varieties were in production and we had no understanding of genetic mechanisms that would contribute to disease resistance. Furthermore, we know even less about the epidemiology and genetic mechanisms for resistance to hop DM. Without significant and rapid progress in developing more economic means to control the disease, U.S. hop production cannot remain competitive in global hop markets, farm profitability will severely decline, and reliance of brewers on foreign hop imports will increase. At the same time, it also is important to identify specific compounds in hop cones that contribute superior flavor attributes, and develop varieties that produce enhanced levels of these compounds for commercial production. The objectives of this research are: 1) Identify, sequence, map and characterize hop genes that confer specific phenotypic
functions of economic importance. 2) Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection. 3) Identify and develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. To solve these problems, both cultivated and wild hop germplasm are being evaluated for their potential use in development of new varieties, traditional plant breeding is being used to incorporate traits that provide disease resistance and superior brewing characteristics into enhanced germplasm, and molecular markers are being developed to decrease the length of time required to develop new varieties. This research addresses objectives of National Programs #301- Plant Genetic Resources, Genomics, and Genetic Improvement; National Program #303 - Plant Diseases; and National Program #306 -Quality and Utilization of Agricultural Products. This work is relevant to U.S. breweries, hop marketing companies, and hop producers and
critical to maintain U.S. leadership in hop production and export is critical with more than 60% of the U.S. hop acreage planted in USDA-ARS varieties. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY2003) Make Genomic Libraries Estimate Genetic diversity/ Publish results Make year 2003 Crosses Year 2 (FY2004) Run micro-satellites/Take field data-1st yr Collect second year data estimating heritability of specific traits Year 3 (FY2005) Finish sequencing genes/ analyze data for Objective 1 Finish micro-satellites/ 2nd year data collection Collect 1st year data and release superior germplasm from year 2001 crosses Year 4 (FY2006) Collect 2nd year data for Objective 2 Collect 2nd year data for Objective 3 Year 5 (FY2007) Release superior germplasm from year 2003 crosses 4a List the single most significant research accomplishment during FY 2006. Molecular Detection of Airborne Spores of the Hop DM Pathogen. The USDA- ARS Forage Seed and
Cereal Research Unit in collaboration with Washington State University developed molecular assays to detect the hop DM pathogen in near real-time from the air of hop yards. This is significant because fungicides commonly are over-used for management of hop DM because rapid and sensitive methods for detecting the pathogen are not available. The assay and a novel spore trapping method were developed and validated under field conditions, and were able to detect airborne spores of the pathogen 10 days before symptoms development. This discovery may allow growers to minimize pesticide use by eliminating applications when the pathogen is not detected, and result in optimized fungicide use, reduced input costs for producers, and enhanced environmental protection. This accomplishment addresses National Program 303 Plant Diseases; Component I - Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens; Problem Statement b) Detection, Identification,
Characterization, and Classification of Pathogens. 4b List other significant research accomplishment(s), if any. Plant Resistance Genes and General Stress-related Genes Cloned and Identified in Hop. The USDA-ARS Forage Seed and Cereal Research Unit identified 221 gene sequences (ESTs) expressed by hop during initial PM infection phase. Hop PM is a recently introduced fungal pathogen causing significant damage on susceptible varieties with virtually no information available on host-pathogen resistance mechanisms. To accomplish this, we used suppressive subtraction hybridization technique that effectively eliminated non-disease resistance genes and purified and amplified plant resistance genes expressed during PM infection. This is the first study of such magnitude identifying genes involved in plant resistance in hop and will ultimately provide geneticists and breeders molecular tools and genetic knowledge to select for resistance to new races of PM as they arise in hop production.
This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component II -Crop Informatics, Genomics, and Genetic Analyses; Problem Statement c) Genetic Analyses and Mapping of Important Traits. 4d Progress report. Opportunities to submit additional programmatic information: A CRADA is in the process of being established with Roy Farms Inc (Yakima, WA) working towards the mutual development of dwarf hop lines. In addition, a joint USA-Czech Republic research project is being pursued to work on improving genetic diversity of hop using plant collection and evaluation of new material. 5. Describe the major accomplishments to date and their predicted or actual impact. Genetic diversity of USA hop germplasm estimated and genetic bottlenecks identified. AFLP Fingerprinting and estimation of genetic diversity of the USDA-ARS germplasm pool identified specific genetically diverse male- female pairs for germplasm development and helped
delineate a significant genetic bottleneck present in current USA hop cultivars. Hop is a clonally propagated perennial crop with virtually no information regarding the genetic relatedness of cultivated and wild accessions present in the USDA-ARS hop germplasm collection. We analyzed the AFLP fingerprints of 154 cultivated and wild North American hop accessions and determined the genetic relationship amongst male-female pairs of accessions. Use of genetic diversity information has enabled better decisions regarding choice of parents for germplasm development and should result in less dependence upon a genetically similar group of cultivars all of which are potentially susceptible to any newly introduced pathogen. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component I Plant and Microbial Genetic Resource Management; Problem Statement b) Assess the Systematic Relationships and Genetic Diversity of Crop Genetic
Resources. Molecular markers to select for resistance to PM identified. PCR techniques including AFLP and differential display analysis were used to identify 24 different potential molecular markers for PM resistance. Conventional breeding techniques for resistance to PM is both time- consuming and prone to errors while selection using molecular techniques helps overcome these shortcomings. We used both differential display on treated and untreated hop lines and AFLP on seven near-isogenic hop accessions (bred for the presence of the seven different R-genes for PM) to identify potential molecular markers for selection. These studies were the first preliminary work identifying genetic sequences in hop involved in plant resistance mechanisms. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component II - Crop Informatics, Genomics, and Genetic Analyses; Problem Statement c)Genetic Analyses and Mapping of Important
Traits. Molecular markers to select for DM resistance identified. Five AFLP molecular markers linked significantly to DM resistance in hop were identified by researchers at the Forage Seed and Cereal Research Unit in Corvallis, OR. Selection for hop DM resistance has defied conventional breeding techniques due to a combination of complex inheritance and environmental influences, and use of molecular breeding techniques may help overcome these difficulties. Representative accessions of the USDA- ARS hop germplasm collection were scored for resistance to DM and molecular markers from AFLP analysis on these same individuals were statistically associated with disease scores using logic regression. These findings will potentially enable the use of molecular selection procedures and increase the efficiency and accuracy of selection for plant resistance to this economically important plant pathogen. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and
Genetic Improvement; Component II - Crop Informatics, Genomics, and Genetic Analyses; Problem Statement c)Genetic Analyses and Mapping of Important Traits. New public hop varieties developed and released. Hop varieties Newport, Santiam, Sterling, Horizon, Bittergold and Teamaker were released in direct response to requests from industry for aroma and bittering hop varieties possessing high yields, better brewing characteristics and/or superior disease resistance. Hop industry representatives requested development of superior, higher-yielding, disease resistant public varieties that would enable growers to reduce costs for production and simultaneously reduce environmental impact by decreasing pesticide usage. Hop varieties were developed in cooperation between USDA ARS Forage Seed and Cereal Research Unit and Washington State University using traditional breeding techniques and additional funding from public and private stakeholders. The development and release of these varieties
should result in significantly higher farm profits due to increased yields coupled with lower costs associated with reduced pesticide applications and will ultimately reduce the impact of pesticides upon our environment. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component III - Genetic Improvement of Crops; Problem Statement c) Germplasm Enhancement/Release of Improved Genetic Resources and Varieties. Hop PM resistance genes cascaded into a single genetic background. The hop PM resistance genes from three different sources of resistance were incorporated into a single genetic background. Hop PM is an obligate fungal pathogen capable of quickly overcoming single-gene based resistance mechanisms and experience in other crops demonstrated extended resistance to similar pathogens using gene cascading techniques. We utilized traditional breeding methods to incorporate genes from three different sources of resistance
into a single genetic background. Our germplasm development efforts should result in extended resistance before the pathogen overcomes plant resistance based upon presence of all three resistance genes. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component III - Genetic Improvement of Crops; Problem Statement c) Germplasm Enhancement/Release of Improved Genetic Resources and Varieties. Fungal resistance gene isolated and inserted into susceptible hop line. In collaboration with Huell Hop Research Center, Germany, we successfully inserted the chitinase gene isolated and identified by our lab in 1999 into a PM-susceptible hop variety resulting in increased resistance to PM. Breweries desired a genetic solution to develop new hop varieties that differ from currently used varieties solely by possession of superior resistance to fungal pathogens. Agrobacterium tumefaciens constructs were used to insert a
continuous-expression-promoter coupled with the hop chitinase gene into a PM susceptible hop variety. The over-expression of this hop chitinase gene appears to result in increased resistance to PM and if proven, offers a solution for developing brewery acceptable hop varieties differing only in the presence of pathogen resistance genes. This accomplishment addresses National Program 301 Plant Genetic Resources, Genomics, and Genetic Improvement; Component III - Genetic Improvement of Crops; Problem Statement c) Germplasm Enhancement/Release of Improved Genetic Resources and Varieties. Leaf wetness reduces PM severity. In collaboration with USDA-ARS Horticultural Crops Research Laboratory, we demonstrated that leaf wetness greatly reduces the severity of infection of hop by its PM pathogen. Infection risk models for hop PM are not accurate in regions with high rainfall because knowledge of the role of leaf wetness on disease is incomplete. Controlled environment experiments
simulated field conditions and found that short durations of surface wetness suppressed the disease. Knowledge of this environmental factor should lead to better prediction of PM diseases since leaf wetness is not considered in current risk assessment models. This accomplishment addresses National Program 303 Plant Diseases; Component II - Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors; Problem Statement c) Population Dynamics, Spread, and Epidemiology of Pathogens. Fungal resistance to phenylamide persists after 20 years. In collaboration with Washington State University, discovered resistance to phenylamide fungicides has persisted in the DM pathogen for nearly 20 years in the near absence of their use. Hop DM management relies heavily on chemical inputs, but indiscriminate use of fungicides has resulted in the rapid development of pathogen resistance. Laboratory evaluations of hop DM isolates from the Pacific
Northwest measured pathogen sensitivity to certain fungicides, revealing that insensitive phenotypes are present at a high frequency within the population. This discovery will allow for the elimination of chemical controls to which the pathogen remains insensitive. This accomplishment addresses National Program 303 Plant Diseases, Component I - Disease Diagnosis Detection, Identification and Characterization of Plant Pathogens; Problem Statement b) Detection, Identification, Characterization, and Classification of Pathogens. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 1) Hop research program tours were presented to representatives of Anheuser Busch and Oregon Hop Commission, Washington Hop Commission and to individual hop producers. Most of
the science and technology presented in these tours were focused towards growers and are adaptable immediately, and hold long-term relevance to production. 2) Presented research reports to the "Hop Research Council" discussing recent advances in hop genetics and plant pathology. The science and technology presented are relevant towards the future and are focused primarily upon brewery as well as grower adaptation and usage. 3) Collaborated with the Oregon State University Integrated Plant Protection Center to make the hop powdery mildew risk index available publicly in near-real time for the entire U.S. This technology is available immediately and is only constrained by grower access to internet. 4) Led USA hop breeders (both private and public) in discussions focused upon systematizing reporting of hop varietal release data and delineating the requirements for germplasm and varietal release. The science and technology presented were applicable to geneticists and breeders and is
available for usage immediately in their programs. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Oral or poster presentations to the following organizations: -Hop Research Council (January, 2006 and August, 2006) -Oregon Hop Research Council (July 2006) -Washington Hop Commission Scientific Meeting (January, 2006)
Impacts
(N/A)
Publications
- Hummer, K.E., Henning, J.A. 2005. Proceedings of the first international humulus symposium. Acta Horticulturae.668. Leuven, Belgium:ISHS. p.262.
- Bassil, N.V., Gilmore, B.S., Oliphant, J.M., Henning, J.A., Hummer, K.E. 2005. Genbank-derived microsatellite markers in hops. Acta Horticulturae. 668:47-52.
- Townsend, M.S., Henning, J.A. 2005. Potential Heteroitc groups in hop as determined by aflp analysis. Crop Science. v.45. p. 1901-1907.
- Danilova, T.V., Henning, J.A. 2005. Sex molecular markers reveal differences of y-chromosome molecular structure among hops of european and american origin. Acta Horticulturae. (ISHS) 668:85-92.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The most critical problem facing the U.S. hop industry is the need for hop varieties that provide superior brewing quality and that contain resistance to two serious diseases, powdery and downy mildew. Although downy mildew has been a problem in U.S. hop producing regions for many years, powdery mildew disease was first identified in the Pacific Northwest growing regions of USA in 1997. Downy mildew breeding has not progressed significantly over the past half century due to the highly variable nature of disease expression in the hop plant. The increased costs associated with controlling these two diseases threaten the economic viability of the entire U.S. hop industry. Presently, the only way to control powdery and downy mildew is by growing of resistant varieties or applying expensive fungicides as
many as eight times per season. The costs of these applications can easily exceed the gross receipts for the crop. When powdery mildew first emerged in U.S. hop production fields, only a few resistant varieties were in production and we had no understanding of genetic mechanisms that would contribute to disease resistance. Furthermore, we know even less about the epidemiology of, and genetic mechanisms for resistance to, downy mildew in hop. Without significant and rapid progress in developing economic means to control the disease, U.S. hop production cannot remain economic and competitive in global hop markets, farm profitability will severely decline, and reliance of brewers on foreign hop imports will increase. At the same time, it also is important to identify specific compounds in hop cones that contribute superior flavor and to develop superior varieties that produce enhanced levels of these compounds for commercial production. The objectives of this research are: 1) Identify,
sequence, map and characterize hop genes that confer specific phenotypic functions of economic importance. 2) Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection. 3) Identify and develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. To solve these problems, both cultivated and wild hop germplasm are being evaluated for their potential use in development of new varieties, traditional plant breeding is being used to incorporate traits that provide disease resistance and superior brewing characteristics into new germplasm, and molecular markers are being developed to decrease the length of time required to develop new varieties. This research addresses objectives of National Programs #301, Plant, Microbial and Insect Germplasm, Conservation and Development-Germplasm Enhancement and Manipulation, and National Program #303, Plant Disease Host Plant Resistance to Disease. This work is
relevant to U.S. breweries, hop marketing companies and hop producers and critical to maintain U.S. leadership in hop production and export. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2003) Make Genomic Libraries Estimate Genetic diversity/ Publish results Make year 2003 Crosses Year 2 (FY2004) Run micro-satellites/Take field data-1st yr Collect second year data estimating heritability of specific traits Year 3 (FY2005) Finish sequencing genes/ analyze data for Objective 1 Finish micro-satellites/ 2nd year data collection Collect 1st year data and release superior germplasm from year 2001 crosses Year 4 (FY2006) Collect 2nd year data for Objective 2. Collect 2nd year data for Objective 3 Year 5 (FY2007) Release superior germplasm from year 2003 crosses 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Finish sequencing
genes/ analyze data for Objective 1. Milestone Fully Met 2. Finish micro-satellites/2nd year data collection. Milestone Fully Met 3. Collect 1st year data and release superior germplasm from year 2001 crosses. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY2006: Collect 2nd year data for Objective 2. Collect 2nd year data for Objective 3. FY2007: Release superior germplasm from year 2003 crosses FY2008 (not included in current CRIS Project): Release superior germplasm from year 2004 crosses 4a What was the single most significant accomplishment this past year? Molecular Markers for Hop Downy Mildew Resistance Identified: USDA-ARS Forage Seed and Cereal Research Unit discovered highly significant correlations between five AFLP molecular markers and resistance to hop downy mildew in the USDA-ARS hop germplasm
collection. Traditional breeding methods have resulted in limited genetic gains for hop downy mildew resistance. We utilized AFLP analysis on 100 hop accessions and scored these same accessions for downy mildew resistance over a two-year period in field plots inoculated with downy mildew. This discovery was important because all previous downy mildew scoring methods for identifying resistance involved inaccurate, laborious and expensive field trials. These molecular markers and methods, coupled with the identified genetic materials, will allow more rapid and inexpensive development of downy mildew resistant varieties and result in less need for fungicide applications, reduced input costs for producers, and enhance environmental protection. 4b List other significant accomplishments, if any. Potential Molecular Markers for Hop Powdery Mildew Resistance Identified: The USDA-ARS Forage Seed and Cereal Research Unit and Horticulture Research Unit in Corvallis, OR identified two
molecular markers for hop powdery mildew resistance acting as potential promoter regions involved in transcription under plant stress response. Hop powdery mildew is a recent invasive pathogen that is causing serious quality and production losses and a quick genetic solution involving germplasm development is required to reduce or eliminate the need for numerous fungicide applications. Differential display was used to identify unique AFLP bands presumably associated with resistance to powdery mildew and then isolated, cloned and sequenced 11 of the resulting markers. The results of this study are significant as they provide both molecular markers for selection and also address basic genetic questions on plant response to hop powdery mildew infection. Markers that prove to be linked to powdery mildew resistance will speed up and improve accuracy of selection for powdery mildew resistance and reduce the costs associated with germplasm development. The Role of Leaf Wetness and
Temperature in Hop Powdery Mildew Infection: The USDA-ARS Forage Seed and Cereal Research Unit discovered that leaf wetness greatly reduces the severity of infection of hop by its powdery mildew pathogen. The environmental conditions that impact the occurrence and spread of the hop powdery mildew are poorly understood, limiting the development of accurate risk assessment systems. Controlled environment studies investigated the effect of varying durations of leaf wetness and temperature on severity of powdery mildew infection of hop, revealing that short durations of leaf wetness are sufficient to reduce infection. This finding is significant because it suggests that surface wetness, and not necessarily rain fall as currently thought, is associated with reduced disease severity in certain environments and climates. Knowledge of this previously un-quantified environmental factor should lead to better prediction of powdery mildew diseases since leaf wetness is not considered in
current risk assessment models, and result in less need for fungicide applications, reduced input costs for producers, and enhanced environmental protection. Long-Term Retention of Insensitivity to Fungicides in Downy Mildew Populations: The USDA-ARS Forage Seed and Cereal Research Unit in collaboration with Washington State University discovered that insensitivity to certain fungicides has persisted in the downy mildew pathogen populations for nearly 20 years in the near absence of their use. Hop downy mildew management relies heavily on chemical inputs, but indiscriminate use of fungicides has resulted in the rapid development of pathogen resistance. Laboratory evaluations of hop downy mildew isolates from the Pacific Northwest measured pathogen sensitivity to certain fungicides, revealing that insensitive phenotypes are present at a high frequency within the population. This discovery will allow for the elimination of chemical controls to which the pathogen remains insensitive,
and result in optimized fungicide use, reduced input costs for producers, and enhanced environmental protection. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. AFLP Fingerprinting and estimation of genetic diversity of the USDA-ARS germplasm pool has allowed the identification of specific genetically- diverse male and female parental pairs for heterotic crosses. Through the process of identifying genetically diverse parents, we determined that a significant genetic bottleneck exists in the hop varieties grown in the USA. We analyzed the AFLP fingerprints of 54 recently collected wild North American hop accessions and determined their relationship to advanced lines present in the USDA-ARS germplasm collection. Use of the genetic diversity information will allow for better decisions regarding choice of parents for future germplasm development work. Differential display analysis was used to identify several molecular
markers for PM resistance. Additional work using AFLP analysis among seven near- isogenic hop accessions (bred for the presence of the seven different R- genes for powdery mildew) revealed 24 different molecular markers. Too date, several of these markers have been determined as coding for promoter regions involved in transcription regulation during plant stresses such as response to disease infection. In work focused upon downy mildew resistance, we identified five AFLP molecular markers that are significantly linked to resistance to downy mildew in hop. This work represents the first time a relationship between specific molecular markers and downy mildew resistance in hop has been demonstrated. All of the work identifying molecular markers for disease resistance will enable breeders to utilized marker assisted selection for disease resistancespeeding up germplasm development and reducing costs associated with breeding and for the grower. We released the hop varieties Newport,
Santiam, Sterling, Horizon, Bittergold and Teamaker. Newport is a replacement for the old USDA variety called Galena and it possesses superior levels of bittering acid, higher yields and, more importantly, resistance to both powdery and downy mildews (Galena is susceptible to both diseases). Sterling is a replacement for the German variety, Perle. Superior, higher-yielding, disease resistant public varieties enables growers to reduce costs for production and ultimately allows brewers to produce a less expensive, more competitive product. The hop powdery mildew resistance genes from three different sources of resistance were incorporated into single genetic background via traditional breeding methods. This germplasm development step will hopefully result in long-term resistance as the pathogen must overcome three resistance genes rather than one. In collaboration with Dr. Elizabeth Seignor (Located in Huell, Germany) we successfully inserted the chitinase gene isolated and
identified by our lab in 1999. This gene is thought to provide broad-based resistance to fungal pathogens and is being used as a model system for future genetic modification work. An additional gene from hops was identified and cloned (beta-glucanase) but has not yet been inserted into hop lines. With the recent hiring of the USDA-ARS hop pathologist we have now discovered that leaf wetness greatly reduces the severity of infection of hop by its powdery mildew pathogen. This information greatly enhances our understanding of the powdery mildew pathogen and will be used to further develop a hop powdery mildew infection model. Finally, in collaboration with Washington State University we have discovered that insensitivity to certain fungicides has persisted in downy mildew pathogen populations for nearly 20 years in the near absence of their useunderscoring the importance of judicious use of fungicides and the need for multi-tactic strategies for long-term management of the disease.
. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 1) Hop research program tours were presented to representatives of Anheuser Busch and Miller Brewing Companies, Hop Growers of America and individual hop producers that included discussions about the integrated USDA-ARS greenhouse, field, chemistry analysis and molecular biology research approach in Oregon. 2) Participated in field tours for the Washington Hop Commission and Oregon Hop Commission and presented a lecture on hop production to students at Oregon State University in the Introductory Brewing Science course. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications
below). Gave oral or poster presentations to the following organizations: -Hop Research Council (January, 2005 and August, 2005) -Oregon Hop Research Council (July 2005) -Washington Hop Commission Scientific Meeting (December, 2004)
Impacts
(N/A)
Publications
- Hummer, K.E., Henning, J.A. 2005. Proceedings of the first international humulus symposium. Acta Horticulturae.668. Leuven, Belgium:ISHS. p.262.
- Bassil, N.V., Gilmore, B.S., Oliphant, J.M., Henning, J.A., Hummer, K.E. 2005. Genbank-derived microsatellite markers in hops. Acta Horticulturae. 668:47-52.
- Townsend, M.S., Henning, J.A. 2005. Potential Heteroitc groups in hop as determined by aflp analysis. Crop Science. v.45. p. 1901-1907.
- Danilova, T.V., Henning, J.A. 2005. Sex molecular markers reveal differences of y-chromosome molecular structure among hops of european and american origin. Acta Horticulturae. (ISHS) 668:85-92.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The most critical problem facing the U.S. hop industry is the need for hop varieties that provide superior brewing quality and that contain resistance to two serious diseases, powdery and downy mildew. Although downy mildew has been a problem in U.S. hop producing regions for many years, powdery mildew disease was first identified in the U.S. in 1997. The increased costs associated with controlling this disease threaten the economic viability of the entire U.S. hop industry. Presently, the only way to control powdery mildew is by applying expensive fungicides as many as eight times per season. The costs of these applications can easily exceed the gross receipts for the crop. When this disease emerged in U.S. hop production fields, there were no resistant varieties in production and no understanding of
genetic mechanisms that would contribute to disease resistance. Without significant and rapid progress in developing economic means to control the disease, U.S. hop production cannot remain economic and competitive in global hop markets, farm profitability will severely decline, and reliance of brewers on foreign hop imports will increase. It also is important to identify specific compounds in hop cones that contribute superior flavor and to develop varieties that produce enhanced levels of these compounds for commercial production. The objectives of this research are: 1) Identify, sequence, map and characterize hop gene conferring specific phenotypic functions of economic importance. 2) Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection. 3) Identify and develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. To solve these problems, both cultivated and wild hop germplasm are being
evaluated for their potential use in development of new varieties, traditional plant breeding is being used to incorporate traits that provide disease resistance and superior brewing characteristics into new germplasm, and molecular markers are being developed to decrease the length of time required to develop new varieties. This research addresses objectives of National Programs #301, Plant, Microbial and Insect Germplasm, Conservation and Development-Germplasm Enhancement and Manipulation (70%) and #303, Plant Disease Host Plant Resistance to Disease (30%). This work is relevant to U.S. breweries, hop marketing companies and hop producers and critical to maintain U.S. leadership in hop production and export. 2. List the milestones (indicators of progress) from your Project Plan. This project replaced CRIS project 5358-21000-030-00D effective 3/10/2004 (Normal Progression) approved through the OSQR process. Year 1 (FY2003) Make Genomic Libraries Estimate Genetic diversity/ Publish
results Make year 2003 Crosses Year 2 (FY2004) Run micro-satellites/Take field data-1st yr Collect second year data estimating heritability of specific traits Year 3 (FY2005) Finish sequencing genes/ analyze data for Objective 1 Finish micro-satellites/ 2nd year data collection Collect 1st year data and release superior germplasm from year 2001 crosses Year 4 (FY2006) Collect 2nd year data for Objective 2. Collect 2nd year data for Objective 3 Year 5 (FY2007) Release superior germplasm from year 2003 crosses 3. Milestones: A. The milestones addressed in FY 2004 are also detailed in the Final Annual Report for the previous project 5358-21000-030-00D which was terminated 3/9/2004 (Normal Progression). Milestones that were scheduled to be addressed in 2004: Run micro-satellites/Take field data-1st yr ' This milestone was completed albeit instead of using microsatellites we used AFLP due to inconsistent results with microsatellites and the need for more work on perfecting the technique
in hop. Collect second year data estimating heritability of specific traits. This milestone was completed. Milestones I expect to address over the next 3 years (2005, 2006, and 2007): Year 3 (FY2005) Finish sequencing genes/ analyze data for Objective 1 Finish micro-satellites/ 2nd year data collection Collect 1st year data and release superior germplasm from year 2001 crosses Year 4 (FY2006) Collect 2nd year data for Objective 2. Collect 2nd year data for Objective 3 Year 5 (FY2007) Release superior germplasm from year 2003 crosses 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY2004 year: Techniques that could help hop breeders to eliminate corsses that would result in inbreeding depression are lacking. Scientists in the Forage Seed and Cereal Research Unit, Corvallis, OR. developed molecular marker technologies to identify significant correlations between genetic similarities and pedigree relatedness. The genetic
similarities and pedigree relatedness between 19 economically important hop (female) varieties with each of 82 male experimental accessions using molecular markers were estimated. This information and the documented molecular procedure will enable hop breeders to make critical decisions regarding which males and females to use for germplasm and varietal development. B. Other significant accomplishments: The genetic diversity among 54 recently collected wild North American hop accessions was estimated. The importance of this accomplishment is that 'fingerprints' for each of these wild accessions can be used to determine their relatedness with known USDA hop clusters. Molecular markers identified by AFLP technology were used to differentiate 3 different clusters that were significantly correlated to the collection location. This new knowledge will permit the use of wild American hop accessions to broaden the gene pool of the USDA hop collection from its currently narrow base and
increase our chances for possessing important genes for future pests or environmental conditions. Incorporation of powdery mildew resistance into commercial hop varieties is critical to the economic sustainability of northwest hop production. Scientists in the Forage Seed and Cereal Research Unit, Corvallis, OR. identified molecular markers that aid in the selection of breeding materials among seven near isogenic hop lines that differ primarily in their respective resistance to the seven known races of powdery mildew. Molecular markers unique to a single genotype that represent potential markers to select for powdery mildew resistance were identified. If these markers are correlated with resistance, they will provide a means of seedling selection for powdery mildew resistance, a measure that would significantly speed development of disease resistant varieties. Twenty two unique AFLP bands that appear to be highly correlated with downy mildew resistance in hop were identified. This is
important because this is the first time a relationship between specific molecular markers and downy mildew resistance in hop has been demonstrated. Quantitative trait loci (QTL) analyses were used to identify these markers. If these markers prove to be correlated with field resistance, they will provide a new means to select for resistance to downy mildew, a desirable trait that has been particularly difficult to identify. C. Significant activities that support special target populations: None D. Progress Report Opportunities to submit additional programmatic information: None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project replaced CRIS project 5358-21000-030-00D effective 3/10/2004 (Normal Progression) approved through the OSQR process. Major accomplishments over the life of the project are included here for continuity. The genetic diversity of male and female accessions contained in the USDA-ARS hop
germplasm collection has been characterized and crosses to take advantage of this diversity are planned. These crosses will provide both short- and long-term benefits to the U.S. hop industry. In addition, plant exploration trips have identified wild North American hop accessions that have been incorporated into the collection. Some of these offer potentially new sources of resistance to fungal and insect pests. The genetic diversity amongst these wild North American hop accessions has been determined. Powdery mildew (PM) and downy mildew (DM) resistant hops have been identified and used to develop resistant plants with superior field production and brewing quality traits. The USDA-21736 experimental line was selected for superior resistance to all races of PM AND DM found in Oregon and Washington. USDA-21736 also exhibited higher levels of total bittering acids and yielded more than any other public variety. This accession was released in 2002 as a new variety named 'Newport'.
We also released five other varieties and a new germplasm. Two new accessions are being investigated at the commercial level for potential brewery use. Crosses have been made that were designed to incorporate resistance to 3 different races of powdery mildew. The manner in which bittering and other flavor compounds important to the brewing industry are inherited have been determined and used to select superior genetic lines with acceptable yield and powdery mildew resistance. We also cloned hop fungal chitinases and glucanases genes we believe are involved in powdery mildew resistance, and have identified numerous molecular markers that will prove useful for identifying new sources of powdery mildew AND downy mildew resistance and genetic diversity in world hop varieties. Hop genetic improvement by this project has helped the USA become the largest producer and world exporter of hops. 6. What science and/or technologies have been transferred and to whom? When is the science and/or
technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Hop research program tours were presented to representatives of Anheuser Busch and Miller Brewing Companies, Hop Growers of America and individual hop producers that included discussions about the integrated USDA-ARS greenhouse, field, chemistry analysis and molecular biology research approach in Oregon. Participants in the International Humulus Symposium, held in Corvallis during August 2004, were given a tour of the USDA-ARS Molecular Biology lab, the Chemistry lab and the Hop Field research station. Participated in field tours for the Washington Hop Commission and Oregon Hop Commission and presented a lecture on hop production to students at Oregon State University in the Introductory Brewing Science course. 7. List your most important publications in the popular press and presentations to
organizations and articles written about your work. Gave oral or poster presentations to the following organizations: Hop Research Council (August, 2004) International Soc. for Hort. Science International Humulus Symposium (August, 2004)
Impacts
(N/A)
Publications
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