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ARS Home Pacific West Area Albany, California Western Regional Research Center Crop Improvement and Genetics Research Research





- Xu, Steven
- Gu, Yong
- L Chingcuanco, Debbie
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)
- Product Quality and New Uses (306)

Research Project: New Genetic and Genomics Resources to Improve Wheat Quality and Resilience to Biotic and Abiotic Stresses **Location:
                            Crop Improvement and Genetics Research** 2023 Annual Report **Objectives**
                                

The primary objective of this project is to enhance the grain yield, end-use quality, and pest resilience of wheat by creating novel genetic and genomics resources. The specific objectives and sub-objectives are listed in the following.

Objective 1: Enhance wheat for high yields and resistance to fungal and insect pests through identification and exploitation of genetic variation in primary gene pool.
Sub-objective 1.A: Identify genes conferring resistance to Hessian fly and greenbug as well as tan spot in a sequenced Ae. tauschii panel.
Sub-objective 1.B: Identify and map genes for resistance to stem rust, leaf rust, stripe rust, tan spot and Septoria nodorum blotch in cultivated emmer wheat using association mapping.
Sub-objective 1.C: Identify and map genes conferring resistance to tan spot in hexaploid wheat line PI 277012.
Sub-objective 1.D: Improve yield potential in elite durum and bread wheat germplasm with salinity tolerance.
Sub-objective 1.E: Develop elite breeding lines of durum and bread wheat with enhanced resistance to Fusarium head blight, sawfly, and Hessian fly.

Objective 2:  Discover and develop novel genetic variation in wheat responsible for superior end-use quality.
Sub-objective 2.A: Characterize diversity of gluten protein genes among U.S. wheat lines.
Sub-objective 2.B: Develop molecular markers that are linked to prolamin alleles associated with the end-use quality and immunogenetic potential in wheat.
Sub-objective 2.C: Improve gluten strength of wheat flour through conventional mutation breeding.

Objective 3: Develop hypoallergenic wheat for healthier and safer food and non-food products.
Sub-objective 3.A: Reduce immunogenic potential of wheat flour through conventional mutation breeding.
Sub-objective 3.B: Relate gluten polyprotein composition to physical properties of novel bioproducts for applications in diverse fields.

Objective 4:  Validate genetically identified trait gene candidates by improving genome editing and transformation efficiency in elite wheat cultivars. 
Sub-objective 4.A: Improve transformation and gene editing efficiency in wheat cultivars
Sub-objective 4.B: Reduce immunogenic potential of wheat flour using efficient gene editing technology
Sub-objective 4.C: Validate gene candidates for resistance to Hessian fly and greenbug identified from Ae. tauschii using efficient transformation.

Objective 5:  Develop Triticeae genomic resources for cereal crop improvement. 
Sub-objective 5.A: Develop Ae. markgraffi reference C genome.
Sub-objective 5.B: Develop Triticeae reference E genome.
Sub-objective 5.C: Develop pan-genomes of durum wheat (AABB) and Ae. tauschii (DD), the bread wheat progenitors.

The resources committed to the project include 3,500 sq. ft. of full-equipped laboratory space, 350 sq. ft. of office space, 3500 sq. ft. of greenhouse space, and field spaces for conducting yield, quality, and disease evaluation trials. **Approach**
                                

Objective 1: Three mapping populations of bread wheat, emmer wheat, and Aegilops tauschii that were previously sequenced or genotyped will be evaluated for their resistance to tan spot, Hessian fly, and greenbug. The phenotypic and genotypic data will be used to identify the quantitative trait loci (QTL) and candidate genes controlling the resistance using association or linkage mapping.  Adapted and elite durum and bread wheat germplasm with enhanced grain yield, salinity tolerance, and disease (Fusarium head blight) and insect (Hessian fly and sawfly) resistance will be developed using backcross breeding coupled with phenotypic assessments and marker-assisted selection (MAS).  Objective 2: The novel genetic variation in wheat responsible for superior end-use quality will be identified by using Prolamin Sequencing Capture Array (ProSeq), transcriptome sequencing, and genotyping with molecular markers targeting specific prolamin alleles. New genetic variations in prolamin components of the gluten will also be generated through mutagenesis using chemicals (ethyl methyl sulfonate) or radiation (fast neutron). Objective 3: Several radiation-mutagenized wheat lines that are deficient in glutenin proteins with epitopes responsible for inducing gluten-related disorders have been backcrossed to the wild-type line, Summit. New mutations in the glutenin and gliadin loci generated in Objective 2 will be introgressed into other elite bread and durum wheat varieties using MAS. The reduction in immunogenic potential of the identified mutant lines will be assessed using commercial kits and antibodies for detecting causal agents for immunogenicity. Both electrospinning and solution blow spinning will be used in the production of gluten fibers from wild-type and hypoallergenic wheat lines. Objective 4: The gene transformation efficiency from co-expression of multiple morphogenetic genes will be tested by generating the WOX5-GRF4-GRF1 construct and introducing it into wheat using Agrobacterium-mediated transformation. CRISPR/Cas9-based genome editing system will be used to create mutations that disrupt the expression of wheat genes encoding immunogenic proteins in Butte86 and Summit. The candidate genes for resistance to Hessian fly and greenbug identified from the Ae. tauschii panel in Objective 1 will be validated using improved gene-editing and transformation. Objective 5:  The reference-quality genome assemblies for Ae. markgraffi, Thinopyrum elongatum, three durum wheat lines, and three Ae. tauschii accessions will be developed based on single-molecule real-time (SMRT) sequencing on a PacBio sequel II (HiFi/CCS mode/cell) Platform. The chromosome-scale assemblies from Ae. markgraffi and Th. elongatum will be released as the reference C and E genomes. The individual chromosome-scale assemblies from the three durum lines and three Ae. tauschii accessions will be provided to the Tetraploid Wheat Pangenome Consortium and the Open Wild Wheat Consortium, respectively, for pan-genome analysis with the individual assemblies from other durum lines and Ae. tauschii accessions under international collaborations. **Progress Report**
                                

This is the first report for the new project 2030-21430-015-000D, which began in March 2023, upon completion of project 2030-21430-014-000D. Most progress from FY23 is included in the final report for 2030-21430-014-000D. This report highlights the progress made in launching new experiments for 2030-21430-015-000D. 

Progress toward Objective 1 involved research activities enhancing wheat for high yields and resistance to fungal and insect pests by identifying and exploiting genetic variation in the primary gene pool. In support of Objective 1, research activities are initiated to identify and/or transfer genes for high yields and resistance to major fungal and insect pests from un-adapted wheat germplasm.

In support of Sub-objective 1A, ARS researchers in Albany, California, evaluated a panel of Open Wild Wheat Consortium (OWWC) Aegilops tauschii accessions for their reactions to two isolates Pti2 (race 1) and DW5 (race 5) of tan spot fungus (Pyrenophora tritici-repentis, Ptr). Among 244 accessions tested with Pti2, 189 were resistant or moderately resistant and 55 were susceptible or moderately susceptible to the isolate. Among 268 accessions that were successfully tested with DW5, 237 were resistant or moderately resistant, with only 31 accessions being susceptible or moderately susceptible to the isolate. The data will be subsequently used to identify the candidate genes controlling the resistance using the k-mer based association mapping pipeline.

Progress under Sub-objective 1B has led to the identification of new sources of resistance to tan spot in cultivated emmer wheat. Among 180 emmer accessions tested, a significant portion were found to be resistant to Ptr isolates 86-124 (race 2) (36%), DW5 (16%), Pti2 (16%), and 331-9 (race 3) (8%), with five accessions showing resistance to all isolates. The emmer panel was also infiltrated with necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB.  Among 180 emmer accessions, most were found to be insensitive (score of 0) to those NEs, while a small frequency of accessions was sensitive (scores between 0.5-3) to Ptr ToxA (2.2%) and Ptr ToxB (4.0%). Genome-wide association studies (GWAS) were performed using each disease trait and genotypic dataset. A total of 17, 21, 16, and 12 single nucleotide polymorphisms (SNPs) were identified to significantly associate with disease traits of Pti2, 86-124, 331-9, and DW5. Interestingly, a race-nonspecific quantitative trait locus (QTL) might be present at a small genomic region (within 602-690 Mbp) on chromosome 4A that confers resistance to all tested isolates.

Progress on Sub-objective 1C has been made in identifying and mapping genes conferring resistance to tan spot in hexaploid wheat line PI 277012. The Grandin × PI 277012 doubled haploid (DH) population (130 lines) was previously genotyped with SSR markers and the 9K SNP Infinium array. The DH population has been successfully evaluated for reactions to Ptr ToxA and four Ptr isolates Pti2, 86-124, 331-9, and DW5. The phenotypic data are being analyzed and will be subsequently used to identify significant QTL associated with reaction to tan spot using linkage analysis. 

Under Sub-objective 1E ARS researchers developed elite breeding lines of durum and bread wheat with enhanced resistance to Fusarium head blight (FHB), ARS researchers also generated backcross BC1F1 hybrids by backcrossing Chinese wheat landrace ‘Wangshuibai’ (donor of Fhb1, Fhb2, Fhb4, and Fhb5) and a wheat-Th. elongatum 7B/7E introgression line carrying Fhb7 to durum variety ‘ND Riverland’ and hard red spring wheat (HRSW) variety ‘ND Frohberg’. As well, they developed elite durum breeding lines with enhanced resistance to sawfly, a total of 240 BC6F5 lines have been developed by transferring the gene for stem solidness from the durum landrace Golden Ball into six durum varieties (Alkabo, Carpio, Divide, Grenora, Joppa, and Tioga) and five breeding lines (D101073, D08900, Carpio-Cd1, Joppa-Cd1, Divide-Cd1) through six backcrosses. Under Sub-objective 1E, 36 elite solid-stem durum lines were selected for a two-year and two-location trial for evaluating yield and end-use quality. These lines and their durum parental varieties are being grown at two locations (Prosper and Williston) for the 1st year in 2023. Additionally elite hard red winter wheat (HRWW) lines were developed for resistance to Hessian fly, a near-isogenic line (NIL) Newton-H26AB carrying H26A and H26B has been backcrossed to hard red winter wheat HRWW varieties ‘KS Western Star’ and ‘KS Dallas’ and breeding lines IDO1906 and IL14-11312. 

In support of Sub-objective 2C to improve gluten strength of wheat flour through conventional mutation breeding, seeds for lines deficient in high-molecular-weight glutenin subunits (HMW-GS) were increased in the field for quality testing. Preliminary data from quality tests confirms that the HMW-GS proteins encoded by the D-genome make the largest contribution to gluten strength and bread volume. The lesion of the first low-molecular-weight glutenin subunit deficient line identified mapped to the short arm of chromosome 3B with a deletion of about two Mb (Glu-B3-S225E). Several novel lines deficient in gliadins encoded by Gli1 and Gli2 loci were identified and mapped to corresponding chromosome groups. The search for lines with the shortest genomic deletion for each locus is in progress.

Progress towards Sub-objective 3A, which is to reduce the immunogenic potential of wheat flour through conventional mutation breeding, involved crossing low-molecular-weight glutenin subunit deficient line Glu-3B-S225E with the HMW-GS triple mutant, to initiate the development of a gluten-free line. Progress in the development of protocols to test for gluten allergenicity includes the generation of antibodies for an omega gliadin epitope that triggers wheat-dependent exercise-induced anaphylaxis. 

In support of Sub-objective 3B, which strives to relate gluten polyprotein composition to the physical properties of novel bioproducts for applications in diverse fields, a solution blow spraying (SBS) device was set up in the laboratory to test the production of gluten-based fibers. Set-up for an electrospinning device is in progress.

Progress toward Objective 5 involved research activities to develop Triticeae genomic resources for wheat improvement. In support of this objective, PacBio sequence data for Ae. markgraffi C genome (Sub-objective 5A) and Th. elongatum E genome (Sub-objective 5B) have been generated and will be subsequently used to develop high-quality reference C and E genomes, respectively. Under Sub-objective 5C, PacBio sequence data for three Ae. tauschii (TOWWC0131, PI 268210, and RL 5271) have been generated and are currently being used in pan-genome analysis with 41 other accessions. **Accomplishments**


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