Progress 02/08/06 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Overall objective is to discover and develop basic knowledge of biological processes in barley that influence the expression of value- added traits and the quality of economically valuable crop tissues. This work includes the identification of chemical signatures of high quality malts by metabolic profiling malts produced from elite barley cultivars and core breeding populations, determination of the contributions of an enzyme to the production of fermentable sugars during industrial processing, and assessment of allelic variation in key enzymes that convert starch to fermentable sugars. Approach (from AD-416) Metabolic profiling of malts will be conducted using standard methods of derivatization, gas chromatography and mass spectral detection/ identification of sample components followed by multivariate analysis of data to identify metabolites that best define the trait "malting quality". Metabolite identification will be done using several mass spectra databases including the 7th edition of Wiley, Essential Oils/Flavor and Fragrance and my lab's personal database. Examination of enzyme contributions to fermentable sugar production will be by use of an immunological probe and by characterizing the recombinant enzyme in a heterologous expression system. Assessment of allelic variation of carbohydrases will be via PCR amplification of expressed mRNA followed by sequence analysis and exploration of functional consequences of documented polymorphisms. This is a final report for project 3655-21000-041-00D to be succeeded by project 3655-21000-052-00D. Substantial results were realized over the five years of the project in the area of reserve carbohydrate metabolism in barley seeds as it relates to the yield of fermentable sugars during germination, malting or the industrial process of mashing. This research expanded upon our previous demonstration of limitations in the enzymatic conversion of reserve carbohydrate to fermentable sugars by identifying genetic variations responsible for these limitations and demonstrating the utility of this information to germplasm enhancement programs in the United States. Using a variety of core collections of barley germplasm relevant to North American breeders we established the presence of exploitable phenotypic variation in two carbohydrases and proved one to be a useful target for germplasm enhancement with molecular marker tools. This work demonstrated that the second carbohydrase is not a candidate for selection using the molecular tools we now have, which are deployed with varying degrees of success in other parts of the world. The likely reason for this global difference is that the germplasm used in the U.S. has an extremely narrow genetic base and has been heavily selected for a malting quality parameter that this carbohydrase is a major determinant of for almost three-quarters of a century resulting in little genetic variation in that particular locus. This work also establishes that portion(s) of the genome outside of this locus are responsible for the genetic component of the observed phenotypic variation in this trait, thereby providing a new avenue for future research. During this five year project the research team has incorporated metabolic profiling as a new research tool which has led to the identification of a number of metabolites potentially of significant interest that were previously unappreciated. Their contribution to overall malting quality will continue to be researched. A method to cheaply and rapidly quantify the entire suite of metabolites was introduced allowing us to monitor the concentrations of all metabolites in solution throughout the malting and mashing process. This method is being evaluated for its ability to distinguish among malts of varying quality. The data collected thus far support this technique�s use as a new measure of malting quality. Accomplishments 01 Determination of utility of beta-amylase intronIII sequence variation in North American malting barley breeding programs. Malting barleys developed in Europe, Asia and Australia had specific variations in the beta-amylase gene sequence that appear to impart functional consequences to the gene product, making sequence variation useful as a molecular marker when selecting for enhanced malting quality. The U.S. malting barley germplasm has been developed with a very narrow genetic basis and has undergone rigorous quality selections for more generations than much of the germplasm used abroad resulting in portions of the genome already being fixed for various quality parameters. We demonstrated no association between the genotype and phenotype of beta-amylase in nurseries from four North American barley breeders and, therefore, we conclude that intronIII sequences are not useful markers of the enzyme�s activity or thermostability in North American malting barleys in use tod The impact is that no resources will be needlessly deployed to incorporate this putative molecular marker as a tool by North American barley breeders unless they knowingly introduce new variation from alien germplasm that has an association between this marker and phenotype already demonstrated. 02 Determination of the utility of osmolyte concentrations as a measure of wort quality. Traditional malt quality criteria are useful in determinin the quality of malt but not as useful in predicting the brewhouse performance defined as the malt's production of a high quality wort. ARS researchers at Madison, Wisconsin, in collaboration with University of Wisconsin researchers demonstrated that wort molarity as measured with a vapor pressure osmometer is better able to distinguish differences in quality among worts made from the most elite germplasm in commercial use today in North American breweries. This provides maltsters, brewers and associated researchers with a new, low cost tool for monitoring wort quality during production or during development of modified brewing methods to meet the needs of specialty brews.
Impacts
(N/A)
Publications
- Duke, S.H., Henson, C.A. 2010. A Comparison of Barley Malt Osmolyte Concentrations and Standard Malt Quality Measurements as Indicators of Barley Malt Amylolytic Enzyme Activities. Journal of American Society of Brewing Chemists. 67(4):206-216.
- Filichkin, T.P., Vinje, M.A., Budde, A.D., Corey, A.E., Duke, S.H., Gallagher, L., Helgesson, J., Henson, C.A., Obert, D.E., Ohm, J.B., Petrie, S.E., Ross, A.S., Hayes, P.M. 2010. Phenotypic Variation for Diastatic power, �-Amylase Activity, and �-Amylase Thermostability vs. Allelic Variation at the Bmy1 Locus in a Sample of North American Barley Germplasm. Crop Science. 50:826-834.
- Havey, M.J., Raines, S., Henson, C.A. 2009. Genetic Analyses of Carbohydrate Accumulation in Onion. Journal of the American Society for Horticultural Science. 134:618-623.
- Vinje, M.A., Duke, S.H., Henson, C.A. 2010. Utilization of Different Bmy1 Intron III Alleles for Predicting �-Amylase Activity and Thermostability in Wild and Cultivated Barley. Plant Molecular Biology Reporter. 28:491- 501.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Overall objective is to discover and develop basic knowledge of biological processes in barley that influence the expression of value- added traits and the quality of economically valuable crop tissues. This work includes the identification of chemical signatures of high quality malts by metabolic profiling malts produced from elite barley cultivars and core breeding populations, determination of the contributions of an enzyme to the production of fermentable sugars during industrial processing, and assessment of allelic variation in key enzymes that convert starch to fermentable sugars. Approach (from AD-416) Metabolic profiling of malts will be conducted using standard methods of derivatization, gas chromatography and mass spectral detection/ identification of sample components followed by multivariate analysis of data to identify metabolites that best define the trait "malting quality". Metabolite identification will be done using several mass spectra databases including the 7th edition of Wiley, Essential Oils/Flavor and Fragrance and my lab's personal database. Examination of enzyme contributions to fermentable sugar production will be by use of an immunological probe and by characterizing the recombinant enzyme in a heterologous expression system. Assessment of allelic variation of carbohydrases will be via PCR amplification of expressed mRNA followed by sequence analysis and exploration of functional consequences of documented polymorphisms. Significant Activities that Support Special Target Populations We previously reported variation in intron structure of a barley carbohydrase (�-amylase) important in the production of fermentable sugars during the malting/brewing process. We have now completed characterizing the intron III alleles in barley germplasm used by North American breeders to develop malting barley cultivars and showed the presence of only four alleles. We have also determined the 5 known SNPs in exons of these genotypes and the activities and thermostabilities of this enzyme from the same genotypes. Additionally, we sequenced and analyzed the promoters from a representative of each of the four allele categories. We have also determined the thermostabilities and activities of �-amylases in a population of barley segregating for winter hardiness and malting quality. We received tissues for metabolic profiling under highly controlled greenhouse conditions for disease progression studies and tissues generated under cold hardening conditions for winter hardiness studies in both malting barley and in oats. We developed specific tissue processing, derivatization and separation methods for oats and refined those used for barley. These three profiling projects are in varying stages of analyses with two of them progressed to the point where peak matrices templates have been built. We extended the studies of malt osmolyte concentrations by examining the relationship between wort osmolyte concentrations, wort sugars, and wort carbohydrase activities and thermostabilities.
Impacts
(N/A)
Publications
- Duke, S.H., Henson, C.A. 2009. A Comparison of Barley Malt Amylolytic Enzyme Activities and Malt Sugar Concentrations. Journal of American Society of Brewing Chemists. 67:99-111.
- Duke, S.H., Henson, C.A. 2008. A comparison of barley malt quality measurements and malt sugar concentrations. Journal of American Society of Brewing Chemists. 66(3):151-161.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Overall objective is to discover and develop basic knowledge of biological processes in barley that influence the expression of value- added traits and the quality of economically valuable crop tissues. This work includes the identification of chemical signatures of high quality malts by metabolic profiling malts produced from elite barley cultivars and core breeding populations, determination of the contributions of an enzyme to the production of fermentable sugars during industrial processing, and assessment of allelic variation in key enzymes that convert starch to fermentable sugars. Approach (from AD-416) Metabolic profiling of malts will be conducted using standard methods of derivatization, gas chromatography and mass spectral detection/ identification of sample components followed by multivariate analysis of data to identify metabolites that best define the trait "malting quality". Metabolite identification will be done using several mass spectra databases including the 7th edition of Wiley, Essential Oils/Flavor and Fragrance and my lab's personal database. Examination of enzyme contributions to fermentable sugar production will be by use of an immunological probe and by characterizing the recombinant enzyme in a heterologous expression system. Assessment of allelic variation of carbohydrases will be via PCR amplification of expressed mRNA followed by sequence analysis and exploration of functional consequences of documented polymorphisms. Significant Activities that Support Special Target Populations 1) We previously established that Agl1 from one H. spontaneum accession has an unusually high number (>50) of polymorphisms for this gene, which was completely invariant in the H. vulgare cultivars that constitute the core of the �MaltGenes� collection. The functional properties (thermostability, Vmax, Km) of the enzyme encoded by the gene from the wild barley were within the ranges found in these properties of the enzymes from MaltGenes cultivars. Therefore, exploitation of this wild barley accession is not useful for improving glucosidase in barley. 2) Real time PCR studies have been completed allowing assessment of variation in intron structure on expression of transcript of a carbohydrase important in the production of fermentable sugars. We expanded the study to include quantifying the expression of a gene encoding an enzyme with the same substrate specificity but with different tissue and developmental expression. 3) Expanded the study of osmolyte concentration (OC) as an indicator of malting quality by completing studies of OC, the production of fermentable sugars during mashing and malt extract. This research relates to National Program 302 � Plant Biological and Molecular Processes by directly contributing to solving Component 2, Biological Processes that Improve Crop Productivity and Quality, Problem Statement 2A: Understanding Growth and Development and Problem Statement 2C: High-Value Products by providing fundamental and applied information on a value-added trait critical to industry and to growers/producers of the raw material.
Impacts
(N/A)
Publications
- Henson, C.A., Duke, S.H. 2008. A comparison of standard and nonstandard measures of malt quality. Journal of American Society of Brewing Chemists. 66(1):11-19.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Overall objective is to discover and develop basic knowledge of biological processes in barley that influence the expression of value- added traits and the quality of economically valuable crop tissues. This work includes the identification of chemical signatures of high quality malts by metabolic profiling malts produced from elite barley cultivars and core breeding populations, determination of the contributions of an enzyme to the production of fermentable sugars during industrial processing, and assessment of allelic variation in key enzymes that convert starch to fermentable sugars. Approach (from AD-416) Metabolic profiling of malts will be conducted using standard methods of derivatization, gas chromatography and mass spectral detection/ identification of sample components followed by multivariate analysis of data to identify metabolites that best define the trait "malting quality". Metabolite identification will be done using several mass spectra databases including the 7th edition of Wiley, Essential Oils/Flavor and Fragrance and my lab's personal database. Examination of enzyme contributions to fermentable sugar production will be by use of an immunological probe and by characterizing the recombinant enzyme in a heterologous expression system. Assessment of allelic variation of carbohydrases will be via PCR amplification of expressed mRNA followed by sequence analysis and exploration of functional consequences of documented polymorphisms. Significant Activities that Support Special Target Populations 1) The characterization of Agl1 was extended from MaltGenes germplasm, where no sequence variation was found, to include additional germplasm not part of the MaltGenes collection. One H. vulgare spp. spontaneum accession encodes an Agl1 with a very high level of sequence variation and will be targeted for further study of this enzyme�s functional properties. 2) The study of bmy1 sequence variations on the enzyme activity and thermostability has been completed in the MaltGenes germplasm. Real time PCR has been optimized and is now being used to determine if the intron III sequence variations have an affect on the expression levels of bmy1. 3) A breeding population segregating for various malting quality traits including beta-amylase was screened for thermostability, intron III sequences and variation in 5 SNPs previously identified as important in enzyme functioning. 4) Metabolic profiling data collection and PCA have been completed. Accomplishments Osmolyte concentrations as a new measure of malt quality. The osmolyte concentration method developed was quantitative, inexpensive, fast, simple and not influenced by a subjective assessment. It correlates well with some other measures of malt quality yet provides different information from that of malt extract data due to its lack of reliance upon density. For this reason it is a better indicator of malt modification than malt extract. The delivery of a new method of monitoring the process of malting or seed germination provides a useful quality control tool for maltsters and a new scientific measure for seed scientists. National Program 302 - Plant Biological and Molecular Processes. The work proposed directly contributes to solving Component 2, Biological Processes that Improve Crop Productivity and Quality, Problem Statement 2A: Understanding Growth and Development and Problem Statement 2C: High- Value Products of the National Program 302 Action Plan by providing fundamental and applied information on a value-added trait critical to industry and to growers/producers of the raw material. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 7
Impacts
(N/A)
Publications
- Duke, S.H., Henson, C.A. 2007. Green Malt Osmolyte Concentration as an Early Indicator of Finished Malt Quality. Journal of American Society of Brewing Chemists. 65(3):145-150.
- Henson, C.A., Duke, S.H., Schwarz, P., Horsley, R., Karpelenia, C.B. 2007. Barley seed osmolyte concentration as an indicator of preharvest sprouting. Journal of American Society of Brewing Chemists. 65(3):125-128.
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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? This project conducts research under National Action Plan 302 Plant Biological and Molecular Processes. The successful production of cereals with value-added traits depends upon our ability to consistently obtain optimal expression of the desired traits. We cannot yet consistently produce cereal grains that meet the stringent specifications required by the processing industries that use the grain. In part, this is due to an incomplete knowledge of the physiology and biochemistry of cereals. Additional limitations result from the ill-defined and changing needs of specific industries as well as imprecise measurements of these qualities. We are working to complete our understanding of the biochemical and physiological processes that result in the conversion of starch to sugars, which is a
crucial value-added trait for malting barleys. Research from this program will potentially lead to development of new or improved crop products, and it will define the properties of value-added traits in such a way that better measurements of quality result. New, more accurate measurements of malt quality will provide the basis for improved pricing of barleys shown to have high malting quality for customers. Growers who lose an anticipated premium (typically $0.25-1. 00/ bushel) for malting quality barley consider crop rejection by malting/brewing industries to be a significant loss. The problem of inconsistent quality is quite serious to industrial users, as unexpected variation in the raw product (malt) requires additional expenses in downstream processing. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) 1. Isolate RNA, synthesize cDNA, sequence appropriate PCR products and analyze sequences from MaltGenes, which is a collection
of important breeding lines used to develop malting barleys used in North America. 2. Re-establish use of the Kakefuda and Duke (1984) gels in lab and confirm use of glycogen as substrate, conduct mashing experiment in triplicate and analyze via native gels. Analyze mashing samples via western blotting. 3. Grow all cultivars at a single location. Year 2 (FY 2007) 1. Clone, express, and evaluate consequences of SNPS in wild type rEnzymes encoded by Agl1. 2. Analyze mashing samples via western blotting. 3. Prepare manuscript. 4. Isolate RNA, synthesize cDNA, sequence appropriate PCR products and analyze sequences from MaltGenes. 5. Malt and conduct standard malt quality analyses; initiate mashing and HPLC analysis to determine RDF values; initiate TMS derivatization and GC- MS metabolite analysis. Year 3 (FY 2008) 1. If SNPs are found that encode proteins with different biochemical characteristics, prepare manuscript. If no SNPs are found in MaltGenes, isolate RNA, synthesize cDNA,
sequence PCR products from 7-10 H. spontaneum accessions. 2. Clone, express, evaluate consequences of SNPs in wild type rEnzymes encoded by Iso1. 3. Complete mashing, HPLC analysis to determine RDF values; TMS derivatization and GC-MS metabolite assays. Year 4 (FY 2009) 1. Clone, express, evaluate consequences of SNPs in wild type rEnzymes encoded by Agl1 from H. spontaneum accessions. 2. If SNPs are found that encode proteins with different thermostabilities, prepare manuscript. If no SNPs are found in MaltGenes , isolate RNA, synthesize cDNA, sequence appropriate PCR products and analyze sequences from 7-10 H. spontaneum accessions. 3. Analyze data. Year 5 (FY 2010) 1. If SNPs are found that encode proteins with different biochemical characteristics in the H. spontaneum accessions, prepare manuscript. 2. Clone, express, evaluate consequences of SNPs in Iso1 from H. spontaneum. Prepare manuscript. 3. Prepare manuscript. 4a List the single most significant research accomplishment
during FY 2006. This research addresses NP 302 Component II Biological Processes that Improve Crop Productivity and Quality, problem statement C) Developing High-Value Products. Evaluation of tools of predicting the value-added trait known as the production of fermentable sugars in malting barleys: The specific accomplishment in this reporting period was the demonstration that primary tool breeders are being encouraged to use to screen their germplasm to increase yield of fermentable sugars during processing is not reliable. The specific problem this accomplishment addresses is the development of tools that increase yield (fermentable sugars) from raw products (cereal grains) and methods that allow accurate prediction of these changes in crop quality and value-added traits. To accomplish this we studied a key enzyme in sugar production in a collection of barley that is used to develop malting cultivars. The impact is that the tool currently being promoted has been rendered less
valuable than was previously thought and has made researchers aware of the need to include more detailed genetic information in addition to short gene sequences of key enzymes to their predictive models. 4b List other significant research accomplishment(s), if any. This research addresses NP 302 Component II Biological Processes that Improve Crop Productivity and Quality, problem statement C) Developing High-Value Products. Evaluation of tools to increase the value-added trait known as the production of fermentable sugars in malting barleys: One specific accomplishment in this reporting period was the demonstration that North American malting barley germplasm contains extremely limited variation in the genetics of one key enzyme in the production of fermentable sugars and that potentially useful variation is present in germplasm collected in the Fertile Crescent of Middle Eastern countries. We identified opportunities to use these non-native barleys as a source of increased enzyme
production that may provide increased yield of product from raw material. Evaluation of methods of predicting the value-added trait known as the production of fermentable sugars in malting barleys: A second specific accomplishment is this reporting period was the development of use of osmolyte concentrations as a measure of and a predictor of malt extract. This provides a novel method for the assessment of the single most important metric of malting barley quality that is used to determine the commercial value of all malting barleys. 5. Describe the major accomplishments to date and their predicted or actual impact. This research addresses NP 302 Component II Biological Processes that Improve Crop Productivity and Quality, problem statement C) Developing High-Value Products. Evaluation of tools of predicting the value-added trait known as the production of fermentable sugars in malting barleys: The specific accomplishment in this reporting period was the demonstration that primary
tool breeders are being encouraged to use to screen their germplasm to increase yield of fermentable sugars during processing is not reliable. The specific problem this accomplishment addresses is the development of tools that increase yield (fermentable sugars) from raw products (cereal grains) and methods that allow accurate prediction of these changes in crop quality and value-added traits. To accomplish this we studied a key enzyme in sugar production in a collection of barley that is used to develop malting cultivars. The impact is that the tool currently being promoted has been rendered less valuable than was previously thought and has made researchers aware of the need to include genetic information in addition to simple gene sequences of key enzymes to their predictive models. This research addresses NP 302 Component II Biological Processes that Improve Crop Productivity and Quality, problem statement C) Developing High-Value Products. Evaluation of tools to increase the
value-added trait known as the production of fermentable sugars in malting barleys: One specific accomplishment in this reporting period was the demonstration that North American malting barley germplasm contains extremely limited variation in the genetics of key enzymes in the production of fermentable sugars and that potentially useful variation is present in germplasm collected in the Fertile Crescent of Middle Eastern countries. We identified opportunities to use these non-native barleys as a source of increased enzyme production that may provide increased yield of product from raw material. Evaluation of methods of predicting the value-added trait known as the production of fermentable sugars in malting barleys: A second specific accomplishment is this reporting period was the development of use of osmolyte concentrations as a measure of and a predictor of malt extract. This provides a novel method for the assessment of the single most important metric of malting barley quality
that is used to determine the commercial value of all malting barleys. 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? The osmolyte assay method for measure of malt extract has been presented at an international meeting and a manuscript has been accepted for publication in the Journal of the American Society of Brewing Chemists. This journal, which is of limited distribution, is targeted specifically to the audience for which the assay was developed. This is a simple, rapid and non-subjective assay that is quantitative. This assay is expected to supplement the traditional measure of extract by providing novel physical measures of malt components.
Impacts
(N/A)
Publications
- Tanaka, Y., Duke, S., Henson, C.A. 2006. Alpha-glucosidases from the glycoside hydrolase family 31 in germinating seeds and seedling leaves of barley. XXIIIrd International Carbohydrate Symposium, July 23-28, 2006, Whistler, Canada. 2006 CDROM.
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