Location: Cereal Crops Research
2023 Annual Report
Objectives
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.
Approach
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.
Progress Report
This is the final report for this project which terminated in May 2023. See the report for the replacement project, 5090-21430-012-000D, “Harnessing Multi-omics for Augmenting Seed Quality and Stress Tolerance in Barley” for additional information.
Objective 1: The barley mini-core population comprising of 165 spring lines were phenotyped in controlled environment chambers for heat, drought, and combined heat and drought stress during heading stage. This population was genotyped using the 50,000 Single Nucleotide Polymorphism (SNP) arrays. Markers associated with seed yield, root, and shoot biomass traits in response to the three abiotic stresses were identified using genome wide association analysis. Three lines were selected based on seed yield as the key trait for determining lines tolerant to these abiotic stresses.
In parallel experiments, a stress tolerant feed barley variety, Otis, and a stress sensitive malting variety, Golden Promise, were subjected to in-depth transcriptomic analysis to identify gene networks associated with abiotic stress responses. A recombinant inbred line population (192 lines) derived from a cross between Golden Promise and Otis was subjected to drought stress in the greenhouse. Seed yield, root and shoot biomass traits were used for identifying genetic loci associated with drought tolerance in this population. This included Ascorbate peroxidase, and Dirigent genes, that can be utilized in marker assisted selection for identifying abiotic stress tolerant lines in barley breeding programs.
Objective 2: The barley malting variety Conrad is used as a standard in the malt quality analysis lab. Seeds of Conrad were micromalted and sampled at five different stages of the malting process – steeping, 1,3 and 5 days after germination and kilning. RNA and proteins were isolated from micromalted samples, sequenced, and assembled into datasets representing the transcriptome, smallRNAome, degradome, and proteome. The transcriptome and proteome datasets have been released following their publications. The smallRNAome and degradome have been analyzed and will be released soon. In the new research project these multi-omic datasets will be integrated along with the metabolome dataset to develop cogent molecular pathways associated with the malting process.
At a very early stage during the malting process, it was established that there was an increase in the reactive oxygen species (ROS) content. This led to identification of the genes responsible for the higher ROS during malting in barley. A survey of the fully sequenced barley genome to identify the Respiratory Burst Oxidase homologs (RBOHs) led to identification of seven other novel gene family members along with the six reported previously. The expression of the Hordeum vulgare Respiratory Burst Oxidase homolog A/C (HvRBOH A/C) was found to be very high during the steeping stage and consistent with the observed increase in the ROS temporal profile. A RNAi transgenic line with reduced expression of the HvRBOH A/C genes however failed to show any phenotype. It was identified that several other members of the HvRBOH family were upregulated in these mutant lines supporting a genetic compensation mechanism to ensure ROS generation during malting.
Transcriptome analysis of Conrad during various malting stages identified two novel genes, Beta-amylase 2(Bmy2) and Disproportionating enzyme 1 (DPE1). These genes and their respective gene products (enzymes) have great potential to improve mashing sugar profiles. Bmy2 is the first beta-amylase gene found to be expressed during malting and encodes a more thermostable enzyme than the predominant seed beta-amylase (Bmy1). DPE1 encodes an enzyme capable of producing substrate, maltopentaose, from maltotriose, an accumulating sugar that cannot be broken down by either alpha- or beta-amylase, theoretically allowing for an increase in fermentable sugar production during mashing. DPE1 and Bmy2 showed significant differences in expression in thirteen elite malting varieties. Bmy2 and DPE1 are potential novel targets for breeders to improve barley malting quality.
Based on published literature it was hypothesized that the Lys3a regulates the expression of the endosperm-specific Bmy1 gene, and other genes associated with malting quality by altering the methylation patterns. Numerous malting quality genes were downregulated in lys3 mutants including Bmy1 and hordein genes as determined by RNAseq. Upregulation of a beta-glucosidase gene, which encodes an enzyme that catalyzes the breakdown of beta-glucans, explains the low beta-glucan values found in lys3 mutants. The causal mutation of the lys3a was found to be at nucleotide 173 in the Barley Prolamin Binding Factor (BPBF) transcription factor gene and affected the expression of at least 184 genes, many being associated with malting quality. Whole genome bisulfite sequencing of the lys3 mutants and parents found no differences in global methylation patterns at CG, CHG, or CHH methylation islands. The hypothesis that the Bmy1 promoter was hypermethylated in lys3a mutants causing downregulation of Bmy1 gene expression was proved to be incorrect. There were no significant differences in CpG methylation between parents and lys3 mutants in Bmy1 or any of the other 16 malting quality associated genes despite their expression being downregulated in the mutants. This study clearly established that the lys3a mutation does not affect DNA methylation in promoters of genes associated with malting quality.
Objective 3: Malts from U.S. germplasm collections submitted to ARS researchers in Madison, Wisconsin, for malt quality analyses were used to generate worts during routine quality analyses. Samples of approximately 500 individual worts were collected, analyzed for solute concentration and for the quantities of a limited suite of metabolites. These metabolites included one heat stress induced metabolite in addition to the precursor and breakdown product of this metabolite. Significant variation was identified both within and between four breeding populations. Data revealed that although significant variation was present within and between these populations, each population was meeting its targeted malt extract values for some lines with a different combination of metabolites. Populations studied were from barley growing regions in the western, mid-western and eastern United States, resulting in a wide range of environments being evaluated. Dry land plots (drought stressed) had a narrower range of stress induced metabolite than did mid-western grown plots.
One of the bottlenecks in advancing functional genetic research in barley has been attributed to the lack of efficient methods for plant transformation. Only one barley variety, Golden Promise, is currently amenable for transformation. A collaboration between the Wisconsin Crop Innovation Center, ARS researchers in Aberdeen, Idaho, and ARS researchers in Madison, Wisconsin, led to the development of a novel method using barley meristem tissue for transformation. More importantly this method was tested using a modern malting barley variety, Gemcraft. A sunflower transcription factor important for conferring drought tolerance, and Bmy2 overexpression constructs have been introduced into Gemcraft using this transformation method. Evaluation of these transgenic lines will be continued in the new research project.
Accomplishments
1. Heat and drought tolerant barley accession identified. One barley accession from the minicore collection was identified as being tolerant to heat and drought stress under greenhouse conditions. Field evaluations under irrigated and rainfed conditions in two different geographical locations confirmed that this accession was drought tolerant. Furthermore, this accession has also been known to have good malting quality traits. This accession will be valuable for the breeders to develop heat and drought tolerant malting barley varieties suitable for the United States.
2. Novel barley transformation technique. A meristem-based transformation technique for manipulating genes of interest in a modern barley variety is now available through the Wisconsin Crop Innovation Center. This technology is valuable for researchers pursuing functional genetic analysis in barley.
Review Publications
Mahalingam, R., Duhan, N., Kaundal, R., Smertenko, A., Nazarov, T., Bregitzer, P.P. 2022. Heat and drought induced transcriptomic changes in barley varieties with contrasting stress response phenotypes. Frontiers in Plant Science. 13. https://doi.org/10.3389/fpls.2022.1066421.
Ajayi, O.O., Bregitzer, P.P., Esvelt Klos, K.L., Hu, G., Walling, J.G., Mahalingam, R. 2023. QTL mapping of shoot and seed traits impacted by drought in barley using a recombinant inbred line population. BMC Plant Biology. 23. Article 283. https://doi.org/10.1186/s12870-023-04292-x.
