Location: Crop Improvement and Genetics Research
2022 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).
Objective 5: Enhance wheat for high yields and resistance to fungal and insect pests through identification and exploitation of genetic variation in primary gene pool.
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 2022 for project 2030-21430-014-000-D “New Genetic Resources for Breeding Better Wheat and Bioenergy Crops” as well as related subsidiary projects. Progress toward Objective 1 involved research activities developing wheat lines with improved end-use quality including decreased immunogenic potential.
In support of Sub-objective 1B, gene bombardment was used to successfully transform two wheat varieties with genome editing machinery designed to target thioredoxin genes and potentially reduce preharvest sprouting. Potentially damaging mutations in a thioredoxin gene on wheat chromosome 2A were recovered and one regenerant from the Bobwhite wheat variety was determined to contain two 4-bp deletions in the thioredoxin gene coding sequence. Another regenerant from the Zak wheat variety was determined to contain a 1-base pair (bp) insertion in the thioredoxin gene. Progeny from these individuals will be selected for these mutations and to identify transgene free lines for further phenotypic analysis. Further transformations of wheat have utilized the Fielder variety and Agrobacterium-mediated transformation for insertion of gene-editing machinery to target a cytochrome P450 monooxygenase gene involved in seed dormancy that has potential to reduce preharvest sprouting. These transformations have targeted a gene on chromosome 5A, 5B, and 5D involved in the oxidative degradation of the seed dormancy-promoting hormone abscisic acid called ABA 8’-hydroxylase. Many substantive edits have been obtained in the lines, and individuals for homozygous, loss of function ABA 8’-hydroxylase alleles have been identified and used to examine their possible resistance to preharvest spouting under controlled mist spraying environment.
Progress under Sub-objective 1C identified lines homozygous for gliadin genes with fast-neutron radiation (FNR) induced mutations and initiated back-crossing to the Summit wheat variety. Using the exome capture tool developed in Sub-objective 2A, we identified 146 genes coding for prolamin genes in the developing seed of Summit. Ribonucleic acid sequencing (RNAseq) experiment was carried out to identify members of the gliadin gene families missing in the homozygous mutant lines. Additional backcrossing of FNR mutant lines lacking low-molecular weight glutenin subunit genes (LMW-GS) was able to achieve greater than 99% genome identity with the Summit variety. Seeds are being bulked for field planting and quality testing. Seed for high-molecular weight glutenin subunit (HMW-GS) ethyl methansulfonate (EMS) and FNR single and select double mutant lines that were previously backcrossed to achieve greater than 99% genome identity with wild-type progenitor were bulked, planted in the field, and are ready for quality testing. 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 processing quality and reduction of flour health-risk related immunogenic proteins.
Progress on Sub-objective 2A has identified complete sets of wheat prolamin genes from different wheat accessions to understand their genetic diversity and possible association with end-use properties of flour. This utilized a highly efficient targeted DNA capture and long-reading sequencing method developed previously. Analysis of gluten protein gene sequences from different wheat accessions revealed large variations with respect to both gene copy number and repetitive structure. In addition, a double haploid population of 87 lines displaying considerable variation in the end-use properties was genotyped using the same targeted sequencing approach. This genotyping data will be used to understand the genetics of wheat prolamin genes. Furthermore, RNAseq analysis on these double haploid lines was performed to determine the expression of individual prolamin genes during seed development. A comprehensive analysis of the expression and genotyping data will help us better understand how variation of prolamin composition and quantity relates to the end-use properties of wheat flour.
Progress under Sub-objective 2B has led to refinement of the proteomic map of flour from wheat line Butte 86 focusing on the non-gluten protein component that is present in flour in low amounts. Two-dimensional gel electrophoresis was performed on non-gluten components of flour extracted with a dilute salt solution. A total of 57 different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins potentially contributing to wheat allergenicity. The predominant proteins in 18 gel spots were gluten proteins. Some of these also contained non-gluten proteins. Analysis of the salt-soluble proteins from a transgenic line in which omega-1,2 gliadins were eliminated by ribonucleic acid (RNA) interference indicated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that comigrated with several alpha gliadins. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining which wheat proteins should be targeted in future gene editing experiments to reduce this potential.
Collaboration with Korean scientists has resulted in progress on Sub-objective 2C by establishing mass spectroscopy as a viable gel-free proteomic method to evaluate high-molecular-weight glutenin subunits that are important contributors to wheat end-use quality. Thirty-eight different Korean wheat varieties were analyzed using an optimized mass spectroscopy technique. Specific glutenin subunit isoforms could be rapidly identified including several pairs that were previously difficult to distinguish from one another due to their very similar molecular weights. This technique is now being developed as a rapid, high-throughput tool for selecting wheat lines containing desirable combinations of high-molecular-weight glutenin subunits that will have improved baking properties.
Data obtained under Sub-objective 2A has been analyzed under Sub-objective 2D to develop molecular breeding tools. The DNA capture array that was designed also contains oligonucleotide probes that target single copy sequences in prolamin genomic regions. Scrutiny of these single copy sequences from different wheat accessions has identified sequence variation that can serve as targets for an innovative SNP genotyping method, called semi-thermal asymmetric reverse PCR (STARP). The genotyping method offers flexible throughput, simple assay design, low operational costs, and platform compatibility. Unique STARP oligonucleotide primers targeting specific genotypes have been designed and will be tested for specificity and utility.
Under Sub-objective 3A, upon retirement of our collaborator from the University of Saskatchewan, a collaboration with ARS scientists in Pullman, Washington, was established to obtain a better resolution of the genomic regions involved in the development of cold tolerance in wheat.
For the affiliated project to develop Aegilops markgrafii reference C genome relating to Objective 2, the Ae. markgrafii accession S740-69 has been sequenced using single-molecule real-time (SMRT) sequencing on a PacBio sequel II (HiFi/CCS mode/cell) Platform. An initial set of 20 Ae. markgrafii S740-69 samples from plants at different developmental stages have been collected. Total RNAs will be isolated for RNA sequencing. The PacBio long reads and RNA sequencing data will be used to assemble C genome and annotate the genes.
To develop elite durum wheat breeding lines and varieties with solid stem for resistance to sawfly, 240 BC6F3 lines with solid stems were selected by genotyping over 2,000 BC6F2 plants derived from backcrossing the solid stem genes from durum landrace Golden Ball into six North Dakota durum varieties and five breeding lines since 2017. These lines have advanced into BC6F5 generation and will be first tested for stem solidness, yield, and agronomic traits at two locations in a preliminary yield trail (PYT). The lines with yield and quality comparable to the durum varieties will be selected as the elite breeding lines for developing new varieties in durum breeding program.
Accomplishments
1. Genomic resource of a wheat wild relative for crop improvement. Aegilops tauschii, the donor of the D subgenome of hexaploid bread wheat, is an important genetic resource for wheat improvement research. An ARS researcher at Albany, California, in collaboration with scientists at University of California, Davis, further improved the sequence assembly of the Aegilops tauschii genome by closing sequence gaps with Pacific Biosciences log-read sequences and generating two optical maps, and manually annotating disease resistance genes, etc. The advances in this version 5 assembly enhance the utility of the Ae. tauschii genome sequences for wheat genetics, biotechnology, and breeding.
2. New genes provide stem rust resistance in wheat. Wheat stem rust is one of the most destructive diseases of wheat, with potential to cause 100% grain loss during severe epidemics. ARS researchers at Albany, California, and Fargo, North Dakota, in collaboration with scientists in Australia, identified and cloned two new forms (haplotypes) of stem rust resistance gene Sr46 derived from the wheat D-genome progenitor. They designated the original Sr46 haplotypes as Sr46_h1 and two new haplotypes as Sr46_h2 and Sr46_h3, respectively. The three Sr46 haplotypes and their linked molecular markers provide new resource and tools for wheat breeders worldwide to develop new varieties with enhanced stem rust resistance in wheat.
Review Publications
Dong, C., Ponciano, G.P., Huo, N., Gu, Y.Q., Ilut, D., McMahan, C.M. 2021. RNASeq analysis of drought-stressed guayule reveals the role of gene transcription for modulating rubber, resin, and carbohydrate synthesis. Scientific Reports. 11. Article 21610. https://doi.org/10.1038/s41598-021-01026-7.
Yoon, S., Bragg, J.N., Aucar-Yamato, S., Chanbusarakum, L.J., Dluge, K., Cheng, P., Blumwald, E., Gu, Y.Q., Tobias, C.M. 2022. Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3. The Plant Genome. Article e20209. https://doi.org/10.1002/tpg2.20209.
Meng, Y., Varshney, K., Incze, N., Badics, E., Kamran, M., Davies, S., Oppermann, L.M., Magne, K., Dalmais, M., Bendahmane, A., Sibout, R., Vogel, J., Chingcuanco, D.L., Bond, C.S., Soos, V., Gutjahr, C., Waters, M. 2021. KARRIKIN INSENSITIVE2 regulates leaf development, root system architecture and arbuscular-mycorrhizal symbiosis in Brachypodium distachyon. The Plant Journal. 109(6):1559-1574. https://doi.org/10.1111/tpj.15651.
Wang, L., Zhu, T., Rodriguez, J.C., Deal, K.R., Dubcovsky, J., McGuire, P.E., Lux, T., Spannagl, M., Mayer, K.F., Baldrich, P., Meyers, B.C., Huo, N., Gu, Y.Q., Zhou, H., Devos, K.M., Bennetzen, J.L., Unver, T., Budak, H., Gulick, P.J., Galiba, G., Kalapos, B., Nelson, D.R., Li, P., You, F.M., Ming-Cheng, L., Dvorak, J. 2021. Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation. G3, Genes/Genomes/Genetics. 11(12). Article jkab325. https://doi.org/10.1093/g3journal/jkab325.
Gaurav, K., Arora, S., Silva, P., Sanchez-Martinez, J., Horsnell, R., Gao, L., Brar, G., Widrig, V., Raupp, J., Singh, N., Xu, S.S., Brown Guedira, G.L., Faris, J.D., Wulff, B.B., et al. 2021. Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement. Nature Biotechnology. 40:422-431. https://doi.org/10.1038/s41587-021-01058-4.
