Research Project: New Genetic Resources for Breeding Better Wheat and Bioenergy Crops

Location: Crop Improvement and Genetics Research

2020 Annual Report

Objectives
One goal of this five-year research project is to characterize prolamin diversity in several different wheat varieties. For commercial hard red spring wheat cultivars Butte 86 and Summit, allergen- and quality-associated molecular markers for specific prolamin genes will be developed. Deep DNA sequencing of prolamin genes expressed in Butte 86 seeds will enable refinement of its proteomic map and assessment of off-target effects of genome editing on wheat flour. In addition, new germplasm will be developed with reduced levels of pre-harvest sprouting (PHS) and lower immunogenic potential. Other goals are to characterize the genetic mechanisms of cold tolerance in wheat and cuticular wax (CW) variation in switchgrass. Collaborations will be pursued with perennial grass breeders to genotype populations for the purposes of applying genomic selection (GS) and increasing breeding efficiency. Objective 1: Develop novel wheat lines with improved end-use quality and decreased immunogenic potential that can be rapidly deployed into wheat breeding programs. Subobjective 1A: Reduce the immunogenic potential of wheat flour through genome editing. Subobjective 1B: Reduce PHS of a white wheat by decreasing thioredoxin h (Trx h) gene expression in developing seeds. Subobjective 1C: Improve gluten strength and reduce immunogenic potential of wheat flour through conventional mutation breeding. Objective 2: Develop new genomic and proteomic tools to assess variability of genes and proteins involved in flour end-use quality and immunogenic potential of U.S. wheat cultivars. Subobjective 2A: Characterize diversity of gluten protein genes among U.S. wheat lines. Subobjective 2B: Refine proteomic map of Butte 86 flour using new DNA sequence information. Subobjective 2C: Develop gel-free targeted proteomic methods to measure the levels of unique peptides in wheat flour. Subobjective 2D: Develop molecular markers that are linked to gluten strength and/or associated with gluten protein genes with high immunogenic potential. Objective 3: Characterize genetic mechanisms of wheat and bioenergy grasses’ responses to abiotic stress for enhanced crop improvement. Subobjective 3A: Identify genetic factors critical to the development of wheat cold temperature tolerance. Subobjective 3B: Determine extent of natural variation for CW in switchgrass and its association with leaf glaucousness and tolerance to water-stress. Objective 4: Generate novel genomic sequence information for pedigree reconstruction and genomic selection in bioenergy grasses to improve breeding. Subobjective 4A: Use reduced representation sequencing to genotype switchgrass and big bluestem for pedigree inference and to obtain kinship matrices. Subobjective 4B: Determine GS accuracies for seed dormancy, cell wall properties, and winter hardiness and map their Quantitative Trait Loci (QTL).

Approach
Objective 1: Wheat lines with improved end use quality or decreased immunogenic potential will be developed that can be rapidly incorporated into breeding efforts. Targeted genome editing using the clustered regularly-interspaced short palindromic repeats (CRISPR) system will be used to create mutations in wheat genes encoding immunogenic proteins such as omega-5 gliadins, omega-1,2 gliadins and alpha-gliadins. Immunogenic potential of selected lines will be determined using sera from patients with confirmed wheat allergies, celiac disease or non-celiac wheat sensitivity. The targeted genome editing approach will also be used reduce preharvest sprouting in wheat by inactivating thioredoxin genes expressed in developing endosperm. In addition, wheat lines deficient in proteins responsible for dough technological properties and/or for inducing gluten-related disorders will be identified using gel electrophoresis to screen a fast-neutron radiation mutagenized population of ‘Summit’. Lines that lack the targeted proteins will be evaluated for their flour quality and allergenic potential. Objective 2: Develop genomic and proteomic tools to assess variation of genes and proteins involved in flour end-use quality and immunogenicity. Sequence and expression diversity of prolamin and thioredoxin genes in different U.S. wheat cultivars will be determined through targeted sequencing and transcriptome analysis. Bioinformatics analysis will identify structural variations. In depth transcriptome sequencing data will be used to refine a proteomic map of ‘Butte 86’ flour. Allele-specific primer assays targeting prolamin gene regions will be developed to enable marker-assisted selection of wheat lines with differing gluten strength and reduced immunogenic potential. Objective 3: Elucidate genetic mechanisms of wheat and bioenergy grasses’ responses to abiotic stress. The underlying genetic factors involved in wheat cold tolerance will be identified by genetic mapping of three doubled haploid populations that exhibit variability in their ability to survive in cold temperatures. Using transcriptomic data, wheat candidate genes whose expression correlates with cold temperature tolerance will be identified. In switchgrass, mapping and diversity populations have been developed and planted across multiple locations. Measurements of epicuticular wax composition, crystal structure and leaf reflectance will be used to map Quantitative Trait Loci (QTL) and perform genome-wide association studies. Objective 4: Generate novel genomic sequence information for pedigree reconstruction and genomic selection (GS) in bioenergy grasses to improve breeding potential of switchgrass and big bluestem. Pedigree reconstruction will be performed in several experimental populations that will be genotyped using genomic DNA sequencing. Simulations will allow estimation of the number of markers required for accurate pedigree reconstruction. QTL will be identified and GS accuracy determined for seed dormancy, cell wall properties, and winter hardiness. Breeding values will be predicted using the method of ridge regression incorporating population and environment effects.

Progress Report
This report documents substantial progress achieved in 2020 for project 2030-21430-014-00-D “New Genetic Resources for Breeding Better Wheat and Bioenergy Crops” as well as related subsidiary projects. Progress in support of Objective 1 involved research activities toward development of wheat lines with improved end-use quality including decreased immunogenic potential under Sub-objective 1A. Toward this goal, the full complement of gluten protein genes in the U.S. wheat Butte 86 was analyzed in detail to design a gene editing approach to reduce the immunogenic potential of wheat flour. One hundred twenty gluten protein genes were identified using gene capture techniques described below. Of these, 67 encoded full-length proteins. To determine which of the genes should be targeted by gene editing, the numbers and positions of epitopes for celiac disease and the serious food allergy wheat-dependent exercise-induced anaphylaxis were assessed for each protein. These data were considered along with transcriptomic data that provided the relative expression levels of genes encoding each protein. The sequences of proteins within the complex gluten protein families also were compared to provide critical information for the development of targeted mass spectrometry methods for the analysis of edited plants. In support of Sub-objective 1B, efforts to generate gene-edited wheat with improved resistance to preharvest sprouting focused on creation of plant transformation vectors designed to produce knock-out mutations of genes encoding essential redox functions. Previously, subsets of these genes were demonstrated to influence preharvest sprouting in gene-silencing experiments. Three vectors were created that each encode the components necessary to produce specific gene-edited wheat. These vectors were used to produce transformed wheat lines in two wheat varieties. Lines are now being characterized at the sequence level for presence of the desired classes of gene knock-out mutations as well as for inheritance of these mutations in subsequent generations. In related work, another set of vectors was created to produce mutations in genes controlling the grain dormancy hormone abscisic acid in wheat. Several transformed wheat lines have been regenerated from tissue culture and are being characterized for presence of desired mutations. These experiments will test if increased levels of abscisic acid can prevent the undesirable levels of alpha-amylase activity in harvested grain that are associated with low grain quality. For Sub-objective 1C, mutational breeding approaches aimed at improving gluten strength for better baking quality and reducing immunogenic potential have produced high-molecular weight subunit glutenin (HMW-GS) double mutants. These were generated by crosses between homozygous lines with small deleted genomic regions. After a second round of backcrossing these were shown to share at least 99% similarity to the original commercial variety. These new genetic stocks will allow determination of the contributions of individual HMW-GS loci to flour quality from each of the A-, B-, and D-subgenomes of a commercial bread wheat. Related work on low-molecular weight glutenin subunits (LMW-GS), was carried out on lines with deletions in the short arm of chromosome 1B. Genomic DNA was isolated and used to estimate the extent of the missing region using the wheat 90K single nucleotide polymorphism array. Lines that contain the smallest deletions but lack the LMW-GS and linked gamma- and omega-gliadins will be further characterized. For Objective 2, work continued on Sub-objectives 2A, B, and D to assess variability of genes and proteins impacting flour quality and immunogenic potential in U.S. wheat cultivars. Based on our previous analysis of gluten protein gene sequences in the reference wheat cv Chinese Spring, a bead-based DNA target capture tool was successfully designed to enrich genomic DNA containing wheat gluten protein genes. A novel genomics approach that combines target DNA capture and long read sequencing was developed to sequence gluten protein genes in different wheat accessions. The high efficiency and accuracy of this approach was validated using sequenced Chinese Spring and wild emmer wheats as test cases. Subsequently, seventeen wheat lines have been genotyped using this approach. Analysis of gluten protein gene sequences from different wheat accessions revealed large variations with respect to both gene copy number and sequence structures. Immunogenic epitopes that potentially trigger celiac disease and food allergy have also been analyzed in selected wheat accessions. Under Sub-objective 2B, efforts to refine proteomic maps of wheat flour proteins continued. Non-gluten proteins were preferentially extracted from Butte 86 flour with a dilute salt solution, separated by two-dimensional gel electrophoresis and proteins in 172 gel spots were identified by tandem mass spectrometry. The development of a proteomic map of the non-gluten proteins makes it possible to use two-dimensional immunoblotting to identify wheat proteins that trigger immunological responses but may be present in low amounts or obscured by the more abundant gluten proteins in total protein preparations. In addition, non-gluten proteins from Butte 86 transgenic lines missing alpha gliadins or LMW-GS were analyzed by two-dimensional gel electrophoresis combined with tandem mass spectrometry. These studies revealed the identities of minor proteins in two very complex regions of the two-dimensional gels that were obscured by the more abundant gluten proteins. Under Sub-objective 2D, the identification of complete sets of gluten protein genes in different wheat accessions facilitated sequence alignments of gluten protein genes to identify variations that serve as targets for designing primers for an innovative genotyping method, called semi-thermal asymmetric reverse polymerase chain reaction (STARP). The genotyping method offers flexible throughput, simple assay design, low operational costs, and platform compatibility. Unique STARP primers targeting specific genotypes have been designed to test their specificities and utility. The work under Objective 3 focused on the responses of wheat and switchgrass to abiotic stress. As part of Sub-objective 3A, genotyping-by-sequencing genetic data was analyzed to generate refined high-density linkage maps that will be used for quantitative trait mapping with collaborators. This analysis is still ongoing. Sub-objective 3B focused on collaborative efforts to map natural variation in leaf surface wax in switchgrass. This year an association mapping population of approximately 400 individuals was evaluated in four different environments using colorimetric wax assays. Data are being analyzed to determine if diversity for leaf surface wax is associated with specific genomic regions. Previously collected leaf surface wax data on a switchgrass mapping population produced from controlled crosses was analyzed and three significant environmentally responsive loci on separate linkage groups were identified. Differences in total leaf surface wax were primarily due to differences in the amounts of long-chain beta-diketones between the population’s founders. Combining this data with other related datasets on the same population will provide a better understanding of water use efficiency and leaf stress tolerance in switchgrass. In related work that involved a greenhouse study of water-stress, multiple switchgrass lines were grown and monitored through eight weeks of water deficit conditions. Physiological measurements were made throughout the experiment to evaluate plant water status. At the end of the experiment, plants were destroyed to obtain metabolic and genomic information. Data between neo-octoploids and wildtype switchgrass lines were compared to determine if ploidy was a factor in abiotic stress resistance and gene-level expression analysis was performed on wildtype and neo-octoploid plant crown tissue. RNA-sequence analysis was used to compare expression patterns of drought-adaptive genes and prominent pathways. The data demonstrated how crown tissue expression differs under water-stress and well-watered conditions. Identification of differentially expressed genes provided new insight into molecular-level workings in key tissues that will help further studies of drought stress and recovery response in an important biofuel crop. Due to disappointing results that were obtained with pedigree reconstruction in switchgrass and big-bluestem last year, Objective 4 is being reconsidered to support as much as possible switchgrass breeding and selection efforts that are using measures of kinship based on known pedigree data in combination with phenotypic data.

Accomplishments
1. Wheat lines developed with reduced immunogenic potential for celiac disease. The gluten proteins, a complex group of proteins responsible for the unique viscoelastic properties of wheat flour, trigger a chronic autoimmune condition called celiac disease in genetically susceptible individuals. ARS researchers in Albany, California, developed transgenic wheat plants in which genes encoding the alpha gliadins, a complex group of gluten proteins containing immunodominant epitopes for celiac disease, were suppressed using RNA interference. Although only a subset of genes encoding proteins with sequences that trigger celiac disease were targeted, all alpha gliadins were eliminated from the flour. The reactivity of flour proteins from the new lines with sera from patients with celiac disease was tested and showed reduced reactivity relative to the control. In addition, tests that are routinely used to assess the performance of flour in producing wheat-based products demonstrated that the mixing properties of the flour were not altered substantially in the transgenic lines although there was a decrease in dough strength. This work contributes to the efforts of reducing the immunogenic potential of wheat flour.

2. Cloning different types of resistance genes against powdery mildew provides a novel approach to breed broad and long-lasting disease resistance wheat varieties. Wheat powdery mildew disease is one of the most destructive diseases threatening wheat production worldwide. ARS researchers in Albany, California, in collaboration with wheat breeders in China, cloned two powdery mildew disease resistance genes that encode different types of proteins. One is a tandem protein kinase, and the other is a nucleotide binding site and leucine-rich repeat protein. Studies showed that both genes displayed a broad resistance against multiple powdery mildew isolates. The knowledge gained and resources generated in this work should facilitate novel strategies to develop wheat varieties with broad and long-lasting resistance to powdery mildew through conventional breeding or biotechnological approaches.

3. Important genetic loci for leaf surface wax trait were mapped for enhanced abiotic stress tolerance. A waxy covering on the surfaces of plants helps prevent water loss and is also a defensive barrier against excessive heat and UV light damage. In switchgrass the underlying genetic basis for production of this waxy covering was studied using genetic linkage mapping populations, mutations, and evidence from model plant species. ARS researchers in Albany, California, uncovered three genetic regions that control leaf surface wax in a large switchgrass mapping population. Leaf wax was measured in this population in multiple locations and years, thereby quantifying the influence of different environments on this trait. Differences in chemical composition and surface micro-topology of the waxy layer were described for the population’s founders that likely represent environmental adaptation to specific local conditions. This work will enable marker-based selection in switchgrass for a component of abiotic stress tolerance.

4. Predictive accuracy of switchgrass genotypic data is inflated in the presence of population structure. Costs of genotyping switchgrass individuals is far less expensive and faster than acquiring phenotypic data necessary for breeding. By using predictive data modeling with dense genotypic data, higher yielding perennial energy crops such as switchgrass can be developed more efficiently. ARS researchers in Albany, California, and Lincoln, Nebraska, analyzed the predictive ability of genotypic data for cell-wall estimations of sugars, theoretical ethanol yields, and forage digestibility from near infrared spectroscopy data in five sub-populations. When population structure was not accounted for, predictive accuracy was 68% higher than if modeling accounted for population structure using different methods. The findings will encourage breeders to use more accurate prediction models that will not bias their selection methods and will lead to stable increases in switchgrass yields and feedstock quality.

Review Publications
Zhang, Y., Schonhofen, A., Zhang, W., Hegarty, J., Carter, C., Vang, T., Chingcuanco, D.L., Dubcovsky, J. 2020. Contributions of individual and combined Glu-B1x and Glu-B1y high-molecular-weight glutenin subunits to semolina functionality and pasta quality. Journal of Cereal Science. 93. https://doi.org/10.1016/j.jcs.2020.102943.
Woods, D., Dong, Y., Bouche, F., Mayer, K., Varner, L., Ream, T., Thrower, N., Wilkerson, C., Cartwright, A., Sibout, R., Chingcuanco, D.L., Vogel, J., Amasino, R. 2020. Mutations in the predicted DNA polymerase subunit POLD3 result in more rapid flowering of brachypodium distachyon. New Phytologist. 227:1725-1735. https://doi.org/10.1111/nph.16546.
Altenbach, S.B., Chang, H., Rowe, M., Yu, X.B., Simon-Buss, A., Seabourn, B.W., Green, P.H., Alaedini, A. 2020. Reducing the immunogenic potential of wheat flour: silencing of alpha gliadin genes in a U.S. wheat cultivar. Frontiers in Plant Science. 11:20. https://doi.org/10.3389/fpls.2020.00020.
Wang, Y., Hou, N., Johnston, J., Sarreal, C., Jarosh, J., Hughes, A.C., Gu, Y.Q., He, X. 2020. Low prevalence of mobile colistin-resistance in U.S. meat, catfish, poultry and genomic characterization of a mcr-1 positive Escherichia coli strain. Food Control. 118. https://doi.org/10.1016/j.foodcont.2020.107434.
Huo, N., Gu, Y.Q., Mc Cue, K.F., Alabed, D., Thomson, J.G. 2019. Draft genome sequence of Agrobacterium tumefaciens strain 1D1526. Microbiology Resource Announcements. 8(45). https://doi.org/10.1128/MRA.01084-19.
Hotton, S., Kammerzell, M., Chan, R., Hernandez, B.T., Young, H.A., Tobias, C.M., Mckeon, T.A., Brichta, J.L., Thomson, N.J., Thomson, J.G. 2020. Phenotypic examination of camelina sativa (L.) crantz accessions from the USDA – ARS National Genetics Resource Program. Plants. 9(5). https://doi.org/10.3390/plants9050642.
Lu, P., Guo, L., Wang, Z., Li, B., Li, J., Li, Y., Qiu, D., Shi, W., Yang, L., Wang, N., Guo, G., Xie, J., Wu, Q., Chen, Y., Li, M., Zhang, H., Dong, L., Zhang, P., Zhu, K., Yu, D., Zhang, Y., Deal, K., Huo, N., Liu, C., Luo, M., Dvorak, J., Gu, Y.Q., Li, H., Liu, Z. 2020. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nature Communications. 11. https://doi.org/10.1038/s41467-020-14294-0.
Xie, J., Guo, G., Wang, Y., Hu, T., Wang, L., Li, J., Qiu, D., Li, Y., Wu, Q., Lu, P., Chen, Y., Dong, L., Li, M., Zhang, H., Zhang, P., Zhu, K., Li, B., Deal, K., Huo, N., Zhang, Y., Luo, M., Liu, S., Gu, Y.Q., Li, H., Liu, Z. 2020. A rare single nucleotide variant in Pm5e confers powdery mildew resistance in common wheat. New Phytologist. https://doi.org/10.1111/nph.16762.
Chanbusarakum, L.J., Bragg, J.N., Cheng, P.K., Aucar, S., Sarath, G., Palmer, N.A., Edme, S.J., Tobias, C.M. 2020. Gene expression and physiological differences in neo-octoploid switchgrass subjected to drought stress. BioEnergy Research. 13:63-78. https://doi.org/10.1007/s12155-020-10092-0.
Watry, H., Zerkle, A.J., Chingcuanco, D.L. 2020. Modified acid-PAGE method for rapid screening and phenotyping of wheat gliadin mutant lines . MethodsX. 7. https://doi.org/10.1016/j.mex.2020.100858.