Location: Cereal Crops Research2021 Annual Report
Objective 1: Identify and characterize germplasm for barley malt production in suboptimal environmental conditions. Sub-objective 1.1: Barley will be assessed for resilience to combined heat and drought stress. Sub-objective 1.2: Assess the impact of abiotic stress on malting quality. Sub-objective 1.3: SNP genotyping of barley lines using Illumina chips and Genome Wide Association Study (GWAS). Objective 2: Identify molecular networks associated with malting, and functionally characterize known and putative genes with the potential to improve malt quality. Sub-objective 2.1a: Determine the transcriptome and the miRNAs involved in regulating the transcriptome in malting barley. Sub-objective 2.2: Analyze proteome changes during various stages of barley malting. Sub-objective 2.3: Integrate transcriptional, post-transcriptional, and proteomic changes during various stages of malting. Sub-objective 2.4: Functionally characterize the putative malting quality genes Bmy2 and DPE1. Sub-Objective 2.5: Characterize the molecular mechanisms of barley lys3a and determine how its function regulates malting quality genes. Objective 3: Determine biochemical or physiological roles of metabolites in barley and oat. Sub-objective 3.1: Identify abiotic stress-induced seed solutes in malting barley. Sub-objective 3.2: Determine if stress-induced seed solutes function as osmoprotectant molecules to hydrolytic enzymes during mashing.
Objective 1. Accessions from the barley mini-core collection, the Vavilov collection, and selected pre-prohibition and modern elite malting barley cultivars will be grown under optimal and abiotic stress conditions. Evaluation of selected tolerant lines will be for a variety of physical traits including biomass and seed yield, physiological traits such as photosynthesis, transpiration, respiration, stomatal conductance and a variety of malting quality traits including standard metrics of quality plus mashing performance. SNP genotyping of the mini-core collection will aid in GWAS for identification of malting quality and abiotic stress associated QTLs. Objective 2. Changes in the transcriptome, miRNAome and the proteome during malting of selected lines will be evaluated. Omics data from these multiple high throughput platforms will be integrated to develop a systems model of the genetic and biochemical pathways involved in the barley malting process. Genetic confirmation of key genes and proteins associated with malting quality and/or abiotic stress tolerance will be conducted via transformation, CRISPER/Cas or via TILLING populations. Barley lys3.a mutants will be evaluated during grain development to determine the mechanism of action on malting quality genes and to identify the causal gene. Select malting quality genes will be evaluated in modern elite malting cultivars during malting. Objective 3. Stress induced metabolites present in malts and rendered soluble during mashing will be chromatographically separated, then detected and identified by mass spectrometry. Resource spectral databases used for identification will include NIST, Flavor and Fragrances and our in-house authentic compound database. Metabolites identified that are commercially available will be used in relevant concentrations to determine if they affect the activity and thermostability of key enzymes involved in the production of fermentable sugars during high temperature mashing.
Studies to address Objective 1 included subjecting the barley mini-core collection to combined heat and drought stress during the heading stage. Seed yield, shoot weight from stressed plants (heat-replicate 1, drought-replicate 2 and combined stress-replicate 2) and corresponding controls were collected for all the 165 lines. The root biomass of the stressed plants and controls from these experiments have not been completed. This research will enable the identification of germplasm for barley malt production in suboptimal environmental conditions. A second replication of the phenotyping for drought in greenhouse for the recombinant inbred line population (approximately 200 lines) derived from a cross between stress tolerant Otis and sensitive Golden Promise has been completed (root and shoot biomass). The threshing of the harvested heads from these experiments is currently ongoing and progressing slowly due to limited access times caused by the 25% building capacity restrictions. Our proteomics analysis published earlier indicated that barley seeds subjected to malting process were expressing many proteins involved in the stress pathway. This prompted the examination of Reactive Oxygen Species (ROS) during malting and the role of the Respiratory Oxidative Burst Homologs (RBOH) gene family in this process. This study identified that HvRBOHs are strongly induced during malting. Furthermore, genetic compensatory mechanisms were invoked in response to RNAi suppression of highly induced HvRBOHs during malting suggesting the ROS and NADPH oxidase enzyme activity is crucial during the malting process. The expression of disproportionating enzyme 1 (DPE1) during malting was determined in 12 malting barleys that are recommended to U.S. barley growers by the largest stakeholder in the malting barley industry. The 12 malting barleys consisted of four types of malting barley (spring and winter 2-row, spring and winter 6-row). The highest DPE1 gene expression was found in winter 2-row barleys (Endeavor and Wintmalt) followed by 2-row spring barleys (AC Metcalf, Hockett, Conrad) with the exception of AAC synergy and CDC Copeland. The two cultivars with the lowest DPE1 gene expression were Innovation and Thoroughbred, which are both 6-row barleys. These data demonstrate a significant variation in expression of DPE1, which encodes an enzyme capable of producing substrate for both alpha- and beta-amylase theoretically allowing for an increase in fermentable sugar production during mashing. Furthermore, DPE1 protein levels during grain development noticeably increased at 17 days after anthesis coinciding with the peak of DPE1 mRNA levels. DPE1 protein was found to be present in a 2-row and 6-row malting cultivar during malting and appears to be processed into a slightly smaller protein as malting progresses. Studies to address Subobjective 2.5 Goal 2.5a, site-specific bisulfite sequencing was attempted on the Bmy1 promoter in three parents and five putative methylation mutants. This research was approved for time-sensitive research in November and PCR primers were created using previously sequenced Bmy1 promoters from ARS scientists in Madison, Wisconsin, and publicly available sequences. However, after numerous rounds of primer development failures presumably caused by SNPs within the Bmy1 promoter in the eight genotypes of interest a new approach was employed. To circumvent the sequence variability the Bmy1 promoter from all eight genotypes (parents and mutants) were sequenced. This revealed a sampling error that necessitated more sequencing to determine if our seed stock was pure. After multiple rounds of sequencing, it was determined that the seed stocks were pure, but a labeling error was the cause of our sequencing discrepancy. Only ~100bp could be PCR-amplified at a time due to the bisulfite treatment so about 10-13 primer pairs were created for each genotype. Despite genotype specific Bmy1 promoter primers covering the entire promoter amplification of each primer pair to give us full coverage of the promoter was elusive and after multiple attempts a new approach was undertaken to best utilize the limited time allowed on site. Failure to obtain full coverage of the Bmy1 promoter using site-specific bisulfite sequencing was perhaps largely due to the inability to work continuously due to the pandemic/maximize telework status that required limited personnel onsite. In order to circumvent this problem a global approach was implemented consisting of whole genome bisulfite sequence, which will allow ARS scientists to determine the entire genomes methylation status in the eight genotypes from developing endosperms. DNA from all eight genotypes (three parents, three lysa mutants, one lys3b mutant, and one lys3c mutant) have been isolated in triplicate, Illumina whole genome bisulfite sequencing (WGBS) libraries have been created and submitted for sequencing using the Illumina NovaSeq6000 (S4 Flowcell, 4 lanes, 2 x 150 bp). Additionally, RNAseq libraries using the TruSeq Stranded mRNA library have been created and are being sequenced alongside the WGBS libraries. In order to address Subobjective 2.5 Goal 2.5b, a new approach was implemented. Previously the causal gene of the lys3a phenotype was thought to be caused by a mutation in the DEMETER gene but new research has indicated that the causal gene is the Barley Prolamin Binding Factor (BPBF). Therefore, the BPBF gene was sequenced in three parents (Bomi, Bowman, and Sloop) and their lys3a mutants. Additionally, two putatively allelic mutants were sequenced in the background of Bomi (lys3b and lys3c). The causal mutation in the BPBF gene was at nucleotide 173 with wild-type containing an adenine (A) and mutants containing a thymine (T). All three parents had the wild-type 173A nucleotide whereas only the lys3a mutant in the Sloop and Bomi background contained the mutant nucleotide (173T). The lys3a mutant in the Bowman background had the wild-type allele as did the lys3c mutant in the Bomi background. In conclusion the lys3a and lys3c mutants in the Bomi background have the 173T causal mutation and thus are predicted to have hypermethylation in endosperm DNA whereas the three parents and the lsy3a in the Bowman background and the lys3c in the Bomi background are predicted to have wild-type seed methylation pattern (i.e. hypomethylation). The research team completed evaluation of the ability of the most abundant metabolite in mashes to provide protection and/or activate the two most important amylolytic enzymes in a barley mash as was previously demonstrated for both high and low temperature isothermal, low gravity mashing. Modest activation was observed for high concentrations at low mash temperatures although freezing protection was not observed for up to six cycles of freezing and thawing.
Im, H., Henson, C.A. 2021. The impact of barley alpha-glucosidases on mashing and the production of fermentable sugars. Journal of the American Society of Brewing Chemists. https://doi.org/10.1080/03610470.2021.1880222.
Mahalingam, R., Graham, D.L., Walling, J.G. 2021. The barley (Hordeum vulgare ssp. vulgare) Respiratory Burst Oxidase Homolog (HvRBOH) gene family and their plausible role on malting quality. Frontiers in Plant Science. 12. Article 608541. https://doi.org/10.3389/fpls.2021.608541.
Vinje, M.A., Henson, C.A., Duke, S.H., Simmons, C.H., Lee, K., Hall, E., Hirsch, C. 2021. Description and functional analysis of the transcriptome from malting barley. Genomics. 113(5):3310-3324. https://doi.org/10.1016/j.ygeno.2021.07.011.