Location: Crop Improvement and Genetics Research2021 Annual Report
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 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.
This report documents substantial progress achieved in 2021 for project 2030-21430-014-00-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-base pair (bp) deletions in the thioredoxin gene coding sequence. Another regenerant from the Zak wheat variety was determined to contain a 1-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. We have obtained evidence of many different substantive edits in the lines that have been regenerated and are currently evaluating progeny from these individuals for homozygous, loss of function ABA 8’-hydroxylase alleles. 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. 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 resulted in the identification of 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 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 co-migrated 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 single nucleotide polymorphism (SNP) genotyping method, called semi-thermal asymmetric reverse polymerase chain reaction (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. Research on an affiliated project relating to Objective 4 has produced several switchgrass lines with loss of function alleles in its two genes encoding centromeric histone H3. The purpose of these lines is to enable rapid inbred production in switchgrass which is highly desirable from a breeding standpoint. Analysis by flow cytometry and sequencing has demonstrated that they frequently produce progeny with missing chromosomes and, infrequently, progeny with complete genome elimination of one parent. As genome elimination is the rate limiting step in inbred production, this approach has some potential to allow new breeding strategies. A separate, affiliated project involving a collaboration with the University of Tennessee has evaluated several lowland switchgrass populations for heterosis and has identified several genetic regions that are involved in the process.
1. Improved wheat genome sequence advances its application in crop improvement research. Understanding adaptation of plants to long-term climate change and periodic environmental extremes has been limited to well-studied model systems. ARS researchers in Albany, California; Lincoln, Nebraska; Madison, Wisconsin; Griffin, Georgia; and Temple, Texas, collaborated with many other U.S. scientists to sequence the large and complex genome of the polyploid bioenergy crop switchgrass. Analysis of biomass and survival among 732 re-sequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive evidence of climate adaptation. Climate–gene–biomass associations were abundant and varied considerably among deeply diverged gene pools. Gene flow was found to have accelerated climate adaptation during the postglacial colonization of northern habitats. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant sub-genome.
2. Genomic mechanisms of climate adaptation in polyploid bioenergy switchgrass. Understanding adaptation of plants to long-term climate change and periodic environmental extremes has been limited to well-studied model systems. ARS researchers in Albany, California; Lincoln, Nebraska; Madison, Wisconsin; Griffin, Georgia; and Temple, Texas, collaborated with many other U.S. scientists to sequence the large and complex genome of the polyploid bioenergy crop switchgrass. Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive evidence of climate adaptation. Climate–gene–biomass associations were abundant and varied considerably among deeply diverged gene pools. Gene flow was found to have accelerated climate adaptation during the postglacial colonization of northern habitats. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome.
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