Location: Crop Improvement and Genetics Research
2024 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 report documents progress for project 2030-21430-015-000D, titled, “New Genetic and Genomics Resources to Improve Wheat Quality and Resilience to Biotic and Abiotic Stresses”, which started in March 2023.
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 continued on identifying and/or transferring 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 the panel of Open Wild Wheat Consortium (OWWC) Aegilops tauschii accessions for their reactions to six greenbug biotypes (C, E, F, G, I and TX1) and three Hessian fly biotypes (GP, L, and vH13). Among 260 accessions tested, 25-35% of the accessions showed resistant reaction to four greenbug biotypes (C, E, I and TX1), while 13-30% were resistant to three Hessian fly biotypes (GP, L, and vH13). The data were subsequently used to identify the candidate genes controlling the resistance to greenbug and Hessian fly based on genome-wide association studies (GWAS), sequence comparisons between resistant and susceptible accessions, and haplotype analysis. For greenbug resistance, one leading candidate gene has been identified on chromosome 7DL, which has been cloned and introduced into susceptible hexaploid wheat (Fielder) to generate complementary transgenic lines for functional validation. In addition, CRISPR-cas9 knockout construct of the candidate gene for greenbug resistance has been generated and in the process of being introduced into the greenbug resistant hexaploid Texas A&M line (TAM112). Diagnostic marker of the candidate gene has been developed and confirmed in TAM resistant and susceptible lines. For Hessian fly resistance, genome-wide association studies (GWAS) identified three loci on chromosomes 2DL, 3DL, and 6DS. While the candidate genes on chromosome 3DL (our lab) and 6DS (our collaborator) have already been cloned and functionally validated (unpublished), 2DL is a novel locus and the candidate gene will be identified and cloned.
Progress under Sub-objective 1B has led to the identification of new sources of resistance to stem rust in cultivated emmer wheat. A panel of 180 cultivated emmer accessions was previously genotyped using a 9K single nucleotide polymorphism (SNP) Infinium array and genotype by sequencing (GBS) method. The panel was previously also evaluated for seeding reactions to seven races (TTKSK, TRTTF, TTTTF, TPMKC, RKQQC, QTHJC, and MCCFC) of stem rust pathogen. Genome-wide association studies led to the identification of cultivated emmer-derived Sr13 locus on chromosome 6A in the panel, and a total of 42 SNP associated with seven races. Most significant SNPs were located on chromosomes 1B, 2B, 4A, 3B, 4A and 7A. Four SNPs on 1A, 2B, 3B and 6A were associated with two or more races, suggesting their significance in conferring stem rust resistance to multiple races.
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 simple sequence repeat (SSR) markers and the 9K SNP Infinium array. The DH population has been evaluated for reactions to Ptr ToxA and four Ptr isolates Pti2, 86-124, 331-9, and DW5. The phenotypic and genotypic data were used to identify significant QTL associated with reaction to tan spot using linkage analysis. The sensitivity to Ptr ToxA was mapped chromosome arm 5BL, presumably the Tsn1 locus. The locus was shown to underline a susceptibility QTL for races one and two. The genomic region on chromosome 4B harboring Rht-B1 was significantly associated with tan spot resistance for all the races and with the seedling plant length. Other minor quantitative trait loci (QTL) were identified on chromosomes 2D, 5D and 7D with each being associated with specific races.
Under Sub-objective 1E for developing elite breeding lines of durum and bread wheat with enhanced resistance to Fusarium head blight (FHB), the backcross BC3F1 hybrids have been made 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’. For developing elite durum breeding lines with enhanced resistance to sawfly, a total of 240 BC6 derived advanced 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). Under Sub-objective 1E, 36 elite solid-stem durum lines have been selected for a two-year and two-location trial for evaluating yield and end-use quality. These lines and their durum parental varieties were grown at two locations (Prosper and Williston) for the 1st year in 2023. These lines are currently being tested in three locations (Prosper and Langdon, ND; Aberdeen, ID) in 2024. For developing elite hard red winter wheat (HRWW) lines 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’, ‘TAM 204’, and ‘TAM 205’ as well as breeding lines IL06-14262 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. Data from quality tests confirmed that the HMW-GS proteins encoded by the D-genome make the largest contribution to gluten strength and bread volume. Another set of HMW-GS deficient lines were planted in the field for seed increase for a second quality testing. 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 2 Mb (Glu-3B-S225E). Genomic analysis revealed the deletion of most of the Glu3B encoded LMW-GS genes and the Gli1B encoded gliadin genes in Glu-3B-S225E. Several novel lines deficient in gliadins encoded by Gli1 and Gli2 loci were identified and mapped to corresponding chromosome groups. One of the mutant lines, Gli-2A_S241C, with estimated deletion of 2.6 Mb deletion in the short arm of chromosome 6A removed all the gliadin genes in Gli2A locus. Another line, Gli-2A_S288B, with an estimated shorter deletion of 0.877 Mb failed to remove two of the Gli2A encoded alpha gliadin genes. Gli-2B-S094D mutant lines has an estimated deletion of 0.56 Mb in the short arm of chromosome 6B removing most of the encoded alpha gliadins except for two (one of which is a pseudogene) which are located about 19 Mb away. The search for lines with the shortest genomic deletion for each prolamin gene encoding loci is continuing.
Progress towards Sub-objective 3A to reduce the immunogenic potential of wheat flour through conventional mutation breeding: low-molecular-weight glutenin subunit deficient line Glu-3B-S225E was crossed with the HMW-GS triple mutant to initiate the development of a gluten-free line. Prolamin loci specific markers were identified, and primers designed for verification by PCR. 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. A polyclonal antibody against a 9-amino acid peptide encoding an epitope that triggers wheat-dependent exercise induced anaphylaxis (WDEIA) detected the omega-5 gliadin protein encoded in the Gli-1B locus in wild type Summit but was not detected in Glu-3B-S225E, which has a deletion that removed most of the gliadin genes in the Gli-1B locus.
In support of Sub-objective 3B 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. The use of SBS device to generate gluten-based films and fiber was initiated.
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) that were generated in 2023 were used to develop high-quality reference C and E genomes, respectively. A high-quality reference C genome has been assembled and is currently being annotated. Additionally, a reference optical map of the C genome was generated to aid in R gene identification in various C genome introgression lines. Under Sub-objective 5C, PacBio sequence data for three Ae. tauschii accessions (TOWWC0131, PI 268210, and RL 5271) and two tetraploid wheat lines (durum ‘Langdon’ and cultivated emmer accession PI 272527) that were generated in 2023 were used to develop individual chromosome-scale assemblies. These chromosome-scale assemblies are currently being used in pan-genome analysis.
Accomplishments
1. New sources of resistance tan spot and Septoria nodorum blotch in emmer wheat. Tan spot and Septoria nodorum blotch (SNB) are fungal diseases affecting major wheat growing regions worldwide and finding new resistance sources are needed in wheat breeding and production. ARS researchers in Albany, California, and Fargo, North Dakota, in collaboration with scientists at North Dakota State University, evaluated the response of a panel of 180 cultivated emmer wheat lines to four fungal strains causing tan spot and two causing SNB. They identified 8–36% of the cultivated emmer wheat lines showed resistance to four tan spot fungal strains, while 43% and 64% of the lines showed resistance to two SNB fungal strains, respectively. By using genetic analysis, they identified 65 DNA markers that highly correlate with tan spot and SNB diseases, with approximately 30% markers being linked to potentially novel resistance genes. This study demonstrated cultivated emmer wheat as a useful source of tan spot and SNB resistance, and the resistant lines and potentially novel genes identified herein can be utilized to improve modern varieties of durum and bread wheat for resistance to both diseases.
2. New germplasm for improving stem rust resistance in durum and bread wheat. Stem rust is one of the most devastating fungal diseases in durum and bread wheat. Durum and bread wheat germplasm lines carrying unique stem rust resistance genes are crucial for developing new durum and bread wheat cultivars with improved stem rust resistance. ARS researchers in Albany, California, and Fargo, North Dakota, developed and released four durum wheat lines Rusty-KLB, Rusty-14803, Rusty-ST464C1, and CATA1, which carry stem rust resistance gene Sr13 alleles Sr13a, Sr13b, Sr13c, and Sr13d, respectively, in the genetic background of durum line Rusty. These lines are useful for wheat geneticists and pathologists in studying the host-pathogen interactions of wheat stem rust and for wheat breeders in breeding durum and bread wheat with stem rust resistance.
Review Publications
Xu, X., Li, G., Lhamo, D., Carver, B.F., Wulff, B., Gu, Y.Q., Xu, S.S., Armstrong, J.S. 2023. Identification of bird-cherry oat aphid and greenbug resistance sources from Ae. tauschii for wheat improvement. Crop Science. 36(5):2913-2924. https://doi.org/10.1002/csc2.21042.
Karki, M., Robbani, M., Chu, C.N., Xu, S.S., Liu, Z., Yang, S. 2024. The Hessian fly resistance gene HvRHF1 is localized in an NBS-LRR gene cluster in barley. Theoretical and Applied Genetics. 137. Article 71. https://doi.org/10.1007/s00122-024-04581-5.
Sharma, J.S., Che, M., Fetch, T., McCallum, B.D., Xu, S.S., Hiebert, C.W. 2024. Identification of Sr67, a new gene for stem rust resistance in KU168-2 located close to the Sr13 locus in wheat. Theoretical and Applied Genetics. 137. Article 30. https://doi.org/10.1007/s00122-023-04530-8.
Wang, R., Axtman, J., Leng, Y., Salsman, E., Hegstad, J., Fiedler, J.D., Xu, S.S., Zhong, S., Elias, E., Li, X. 2023. Recurrent selection for Fusarium head blight resistance in a durum wheat population. Crop Science. 64(2):617-630. https://doi.org/10.1002/csc2.21179.
Lopez, B., Ceciliato, P., Takahashi, Y., Rangel, F., Salem, E., Kernig, K., Chow, K., Zhang, L., Sidhom, M., Seitz, C., Zheng, T., Sibout, R., Chingcuanco, D.L., Woods, D., McCammon, A., Vogel, J., Schroeder, J. 2024. CO2 response screen in grass Brachypodium reveals the key role of a MAP kinase in CO2-triggered stomatal closure. Plant Physiology. Article kiae262. https://doi.org/10.1093/plphys/kiae262.
Thieme, M., Minadakis, N., Himber, C., Keller, B., Xu, W., Rutowicz, K., Matteolli, C., Bohrer, M., Rymen, B., Chingcuanco, D.L., Vogel, J., Sibout, R., Stritt, C., Blevins, T., Roulin, A. 2024. Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon. PLoS Genetics. 20(3). Article e1011200. https://doi.org/10.1371/journal.pgen.1011200.