Location: Crop Improvement and Genetics Research
2019 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
In support of Sub-objective 1A, transgenic wheat lines developed using Ribonucleic acid (RNA) interference were characterized in detail. Lines were selected in which omega-1,2 gliadins containing immunodominant epitopes in celiac disease (CD) were significantly reduced in flour with few changes in the levels of other proteins. The allergenic potential of the flour was evaluated using antibodies from a collection of CD patients and end-use quality of the flour was assessed. The data demonstrated that elimination of omega-1,2 gliadins resulted in flour with decreased immunoreactivity and improved end-use quality. A second set of experiments focused on alpha gliadins containing immunodominant CD epitopes. Although only a subset of the proteins was targeted, all alpha gliadins were effectively eliminated from the flour in these lines. In addition, there were small reductions in the levels of high molecular weight glutenin subunits (HMW-GS). Mixing studies demonstrated that the overall quality of the flour declined in these lines. Further study of the immunogenic potential of the flour is ongoing. In the last set of experiments, a subset of low molecular weight glutenin subunits (LMW-GS) that contain CD epitopes were targeted. While the desired LMW-GS were eliminated in the transgenic lines, decreases in the levels of all other LMW-GS were also observed. The data demonstrate, that RNA interference can be used to silence entire families of gluten protein genes. However, it is difficult to target specific genes within these complex gene families since very small regions of deoxyribonucleic acid (DNA) sequence identity are sufficient for silencing. Nonetheless, these studies provide a foundation for future clustered regularly interspaced short palindromic repeats (CRISPR) genome editing experiments.
In support of Sub-objective 1B, progress has been made in addressing the susceptibility of wheat to pre-harvest sprouting and managing levels of alpha-amylase in the mature grain to within limits required by bakers and millers. The susceptibility of the spring wheat cultivar ‘Zak’ to preharvest sprouting was confirmed after performing repeated preharvest sprouting experiments in a mist chamber environment. The genome sequences of genes to be targeted in a CRISPR genome-editing approach have been verified in Zak and promising conserved sites in gene copies from the wheat A, B, and D genomes have been identified. Transformation of immature wheat embryos is being initiated with specific editing-enabled constructs.
In support of Sub-objective 1C, we developed methods to rapidly resolve wheat gliadin storage proteins using acid polyacrylamide gel electrophoresis to screen for gliadin gene mutations in a mutagenized commercial variety of bread wheat. Phenotyping of seeds from more than 1000 lines identified 15 omega-gliadin, 10 gamma-gliadin, and eight alpha-gliadin protein deficient mutant lines. Using more traditional protein separation techniques, several mutagenized lines deficient in a low-molecular weight glutenin subunit protein were also identified. We continued a recurrent backcrossing program with several high-molecular weight glutenin subunit deficient mutant lines to create near-isogenic lines and eliminate mutations unlinked to the specific loci of interest. These sets of gluten protein gene mutant lines will allow a better understanding of wheat dough properties and provide genetic tools for improvement of wheat flour quality and possible reduction of flour immunogenicity.
In support of Sub-objective 2A, genomic regions carrying wheat storage protein genes that are members of the prolamin gene family were analyzed in detail in the sequenced genome of hexaploid wheat cv. Chinese Spring. The structural organization, evolution, and expression of individual gene family members were revealed and the complete set of prolamin gene sequence information was retrieved, allowing for more accurate and robust characterization of individual gene expression profiles using RNA-sequencing based analyses. In addition, the sequence information was used to design a tool for allowing unambiguous resequencing from highly repetitive prolamin genes to allow discovery of alleles strongly associated with baking quality and immunogenicity. Furthermore, the corresponding prolamin genomic regions in wild emmer tetraploid wheat were analyzed to help understand the domestication history of wheat and exploit the possible utility of wild emmer diversity for the improvement of hexaploid wheat.
In support of Sub-objective 2B, individual flour proteins from the Chinese Spring cultivar were separated by two-dimensional gel electrophoresis (2-DE) and individual 2-DE spots were analyzed by tandem mass spectrometry to obtain partial peptide sequences. Using this information 72 spots were linked to specific gluten protein gene sequences assembled in Subobjective 2A. Sequences of all gluten proteins were examined for the presence of known epitopes for CD and wheat-dependent exercise-induced anaphylaxis and accumulation levels of proteins containing these epitopes were evaluated. This work provides critical information that will facilitate both breeding and biotechnology approaches aimed at reducing the immunogenicity of wheat flour.
In support of Sub-objective 2C, in collaboration with ARS scientists in Fargo, North Dakota, an innovative Single nucleotide polymorphisms (SNP) genotyping method, called semi-thermal asymmetric reverse PCR (STARP), was adopted in the lab. The genotyping method offers flexible throughput, simple assay design, low operational costs, and platform compatibility. Unique primers targeting prolamin regions based on sequence information were designed to test the specificity of this method for genotyping wheat quality related traits.
In support of Sub-objective 3A, three double haploid mapping populations generated by a collaborator at the University of Saskatchewan, Canada that were previously genotyped using a genotyping-by-sequencing (GBS) method were analyzed in detail. Initial versions of three high-density linkage maps were created for the populations which vary for cold tolerance. These data will be used to obtain a better resolution of the genomic regions involved in the development of cold tolerance in wheat.
In support of Sub-objective 3B, leaf surface wax from 800 switchgrass individuals were measured at four different field locations during the summer growing season of 2018 while collaborators at the University of Texas obtained genome sequence information from the same lines and complementary phenotypic data that will allow the localization of specific genome regions involved in wax deposition. ARS colleagues in Maricopa, Arizona, also assisted with wax chemical analysis, and the wax surface characteristics of the population’s founders were visualized using scanning electron microscopy. This information will lead to a greater understanding of the role of the leaf surface wax in water retention, light scattering, and chemical defense of switchgrass.
Progress toward Objective 4 so far has involved genotyping of big bluestem breeding populations and switchgrass breeding populations established by ARS collaborators in Lincoln, Nebraska, for determining parentage in open pollinated breeding populations. We were able to identify genetic variation from these lines using a variety of techniques and assign genetic similarity scores among related and unrelated individuals. For switchgrass, 1,296 individuals were genotyped altogether, and these genotypes were used to infer pedigrees. In cases where the pedigree was known, the algorithms that were employed correctly assigned parents only 30% of the time. This poor accuracy could be attributed to high levels of kinship among the parents of the crosses, sample or plant mis-identification, or contamination of field material from weeds of the same species that are difficult to identify. In big bluestem, which has six copies of each genome, 416 individuals were genotyped. The accuracy of genotyping was difficult to determine with certainty so two algorithms for detecting genetic variation were used. Relationships among the big bluestem individuals could be assigned a precise value based on genetic similarity, but the population did not provide enough information to positively identify any full siblings or half-siblings among those individuals that were known to have the same maternal parent. Reasons for failure are likely due to the same reasons that were observed with switchgrass. In the absence of a pedigree, the genotypes themselves may be useful for identification of diverse parents or for other breeding techniques, such as genomic selection.
In related work in collaboration with researchers at the University of Tennessee, several switchgrass populations representing about 900 individuals that show differences in heterosis between families are in the process of being genotyped as a preliminary step to performing genetic analysis to identify genomic regions controlling heterosis.
Work begun during a prior project was completed and reported on that demonstrated the degree to which genomic information could be predictive of cell-wall estimations of sugars, ethanol yield, and other constituents from near infrared spectroscopy data. However, the presence of population structure impacted predictive ability and needed to be accounted for in any strategy for molecular breeding of switchgrass.
Accomplishments
1. Wheat lines developed with reduced immunogenic potential for celiac disease and improved end-use quality. Celiac disease is a chronic inflammatory disease triggered by the gluten proteins of wheat, barley and rye in genetically susceptible individuals that affects up to 1.4 percent of the worldwide population. Researchers in Albany, California, developed transgenic wheat plants in which genes encoding the omega-1,2 gliadins, a group of gluten proteins containing immunodominant epitopes for celiac disease, were suppressed using Ribonucleic acid (RNA) interference. Analysis of flour proteins from the transgenic plants by 2-dimensional gel electrophoresis demonstrated that the omega-1,2 gliadins were eliminated from the flour without significant effects on the levels of other flour proteins. Reactivities of flour proteins with antibodies from a collection of celiac patients were reduced significantly in the transgenic lines and end-use quality of the flour was improved. The work demonstrates that molecular approaches can be used to reduce the immunogenic potential of wheat flour without compromising the functional properties that give wheat its commercial value.
2. Release of novel bread wheat germplasm with deficiencies in high-molecular weight glutenin seed storage proteins encoded by the Glu-B1 locus. Genetic variations in the high-molecular weight glutenin seed storage protein loci correlate well with dough quality. Different allelic pair combinations in these genes vary in their effect on dough properties. The gluten proteins, including those high-molecular weight glutenin storage proteins encoded by the Glu-B1 locus are known to contain immunogenic epitopes that cause wheat-related food allergies and celiac disease. Researchers in Albany, California, released a set of five Glu-B1 locus deficient wheat lines. These lines, together with other gluten protein deficient lines, will be used to develop wheat varieties with altered seed protein composition, with modified dough properties, and with reduced immunogenic potential.
3. Sensitizing allergens in a severe food allergy are encoded on two different wheat chromosomes. Omega-5 gliadins are wheat gluten proteins that are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). Researchers in Albany, California, evaluated a mutant wheat line missing the major omega-5 gliadins as a result of a small deletion in chromosome 1B. While the mutant line showed reduced immunoreactivity to sera from WDEIA patients, two minor immunoreactive proteins were detected in both the mutant and wild-type lines. Analysis of the two proteins revealed that both were likely to be encoded by omega-5 gliadin genes on chromosome 1D that previously were thought to be inactive. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.
Review Publications
Altenbach, S.B., Chang, H., Simon-Buss, A., Jang, Y., Denery-Papini, S., Pineau, F., Gu, Y.Q., Huo, N., Lim, S., Kang, C., Lee, J. 2018. Towards reducing the immunogenic potential of wheat flour: Omega gliadins encoded by the D genome of hexaploid may also harbor epitopes for the serious food allergy WDEIA. Biomed Central (BMC) Plant Biology. 18:291. https://doi.org/10.1186/s12870-018-1506-z.
Altenbach, S.B., Chang, H., Yu, X.B., Seabourn, B.W., Green, P.H., Alaedini, A. 2019. Elimination of omega-1,2 gliadins from bread wheat (Triticum aestivum) flour: effects on immunogenic potential and end-use quality. Frontiers in Plant Science. 10:580. https://doi.org/10.3389/fpls.2019.00580.
Fielder, J.D., Lanzatella, C., Edme, S.J., Palmer, N.A., Sarath, G., Mitchell, R., Tobias, C.M. 2018. Genomic prediction accuracy for switchgrass traits related to bioenergy within differentiated populations. Biomed Central (BMC) Plant Biology. 18:142. doi.org/10.1186/s12870-018-1360-z.
Huo, N., Gu, Y.Q., McCue, K.F., Alabed, D., Thomson, J.G. 2019. Complete genome sequence of Agrobacterium fabrum strain 1D159. Microbiology Resource Announcements. 8(19):e00207-19. https://doi.org/10.1128/MRA.00207-19.
Xu, L., Dong, Z., Fang, L., Luo, Y., Wei, Z., Guo, H., Zhang, G., Gu, Y.Q., Coleman-Derr, D.A., Xia, Q., Wang, Y. 2019. OrthoVenn2: a web server for the whole-genome comparison and annotation of orthologous clusters across multiple species. Nucleic Acids Research. 47(W1):W52-W58. https://doi.org/10.1093/nar/gkz333.
Huo, N., Zhu, T., Zhang, S., Mohr, T.J., Luo, M., Lee, J., Distelfeld, A., Altenbach, S.B., Gu, Y.Q. 2019. Rapid evolution of alpha-gliadin gene family revealed by analyzing Gli-2 locus regions of wild emmer wheat. Functional and Integrative Genomics. https://doi.org/10.1007/s10142-019-00686-z.
Ma, G., Zhang, W., Liu, L., Chao, W.S., Gu, Y.Q., Qi, L., Xu, S.S., Cai, X. 2018. Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat. Biomed Central (BMC) Plant Biology. 18:224. https://doi.org/10.1186/s12870-018-1442-y.
Alabed, D.F., Huo, N., Gu, Y.Q., McCue, K.F., Thomson, J.G. 2019. Draft genome sequence of serratia sp. 1D1416. Microbiology Resource Announcements. 8(3):e01354-18. https://doi.org/10.1128/MRA.01354-18.
Blake, V.C., Woodhouse, M.R., Lazo, G.R., Odell, S.G., Wight, C.W., Tinker, N.A., Wang, Y., Gu, Y.Q., Birkett, C.L., Jannink, J., Matthews, D.E., Hane, D.L., Michel, S.L., Yao, E., Sen, T.Z. 2019. GrainGenes: centralized small grain resources and digital platform for geneticists and breeders. Database: The Journal of Biological Databases and Curation. 2019.
Altenbach, S.B., Chang, H., Simon-Buss, A., Mohr, T.J., Huo, N., Gu, Y.Q. 2019. Exploiting the reference genome sequence of hexaploid wheat: a proteomic study of flour proteins from the cultivar Chinese Spring. Functional and Integrative Genomics. 1-16. https://doi.org/10.1007/s10142-019-00694-z.
