Research Project: Genetic Improvement of Small Grains and Characterization of Pathogen Populations

Location: Plant Science Research

2021 Annual Report

Objectives
Objective 1. Identify and develop improved small grain germplasm with resistance to rusts, powdery mildew, Fusarium head blight, necrotrophic pathogens, and tolerance to freezing conditions during winter and spring. Sub-objective 1a. Develop wheat germplasm with resistance to stripe rust, leaf rust, stem rust, and powdery mildew. Sub-objective 1b. Develop wheat germplasm with resistance to Fusarium head blight (FHB). Sub-objective 1c. Develop wheat germplasm with resistance to Stagonospora nodorum blight (SNB). Sub-objective 1d. Identify oat, wheat and barley germplasm with tolerance to freezing. Objective 2. Develop improved methods of marker-assisted selection and genomic prediction, and apply markers in development of small grains cultivars. Sub-objective 2a. Identify and characterize new QTL for important traits in eastern winter wheat germplasm. Sub-objective 2b. Evaluate important traits in eastern winter wheat using molecular markers. Sub-objective 2c. Develop new eastern winter wheat germplasm using marker-assisted breeding and genomic selection. Objective 3. Develop new wheat and barley germplasm and cultivars having enhanced end-use characteristics for the eastern United States. Objective 4. Target resistance breeding efforts accurately by determining the relevant geographic variation in pathogen virulence profiles and the range of mycotoxin potential in pathogen populations. Sub-objective 4a. Determine the virulence frequencies and population structure in the wheat powdery mildew pathogen, Blumeria graminis f. sp. tritici, from different regions in the U.S. Sub-objective 4b. Identify and determine toxicological importance of minority Fusarium species causing FHB of wheat in North Carolina. Objective 5: Speed up breeding winter wheat germplasm with resistance to scab using doubled haploid technology. [NP301, C1, PS1A, PS1B]

Approach
1a. Cross elite, adapted lines with sources of seedling and adult plant resistance to stripe rust, leaf rust, stem rust, and powdery mildew. Coordinate efforts to identify resistant lines in field breeding nurseries evaluated throughout the southeastern United States and in Njoro, Kenya (for Ug99). Evaluation with reliable molecular markers for known resistance genes. 1b. Continue use of inoculated, misted screening nurseries to evaluate regional and in-house breeding materials. Develop, evaluate and refine genomic selection models for scab resistance traits. 1c. Conduct appropriate phenotyping of regional and in-house breeding materials, including mapping populations, in inoculated Stagonospora blight nurseries to assist in locating the genes and associated markers to allow for marker-assisted selection. 1d. Select wheat and oat germplasm with superior resistance to freezing first by identifying genotypic differences in freezing patterns using IR technology. Crosses will be made with adapted cultivars to develop germplasm with improved resistance to freezing conditions. We will continue coordinating an oat and barley uniform nursery. 2a. Use sequencing based genotyping techniques to develop high-density genetic linkage maps of bi-parental mapping populations and association mapping populations as they are developed. Populations are phenotyped in conjunction with other unit scientists to identify regions of the genome involved in resistance to LR, YR, SR, PM, and SNB. 2b. Evaluate diverse germplasm with molecular markers linked to genes for pest resistance, agronomic and end-use quality, determine the level of marker polymorphism and the presence of favorable alleles in breeding lines. 2c. Apply MAS to introgress and pyramid new fungal resistance genes into eastern winter wheat germplasm. Genotype three-way cross and backcross F1s for populations entering into a doubled-haploid (DH) production pipeline. 2c. Use different parameters based on genomic position and linkage disequilibrium to select SNP sets that can be tested via cross-validation to identify the optimum number and most informative markers for GS. 3. Each year, approximately 600 crosses will be made to combine superior quality, yield, agronomic, and disease and insect resistance using recurrent parents from the program, as well as new sources of diversity. Utilize combinations of molecular markers with phenotypic selection and screening to accumulate favorable agronomic traits. Phenotyping and selection for improved hard wheats lines; grow and select populations under organic and conventional conditions. 4a. Samples will be gathered in each of two years from two to five states per region and derive single-pustule isolates; The phenotyping will be done in growth chambers using standard detached-leaf methodology. Population structure will be evaluated using molecular markers. 4b. Scabby wheat spikes will be collected from fields across a broad geographic range of North Carolina. Sequencing of the transcription elongation factor will be used to determine species. Population genetic analyses will determine if the Fusarium species are geographically clustered, or evenly distributed.

Progress Report
In support of Objective 1C, high density genetic linkage maps were analyzed by ARS scientists at Raleigh, North Carolina in conjunction with phenotypic data for soft red winter wheat mapping populations. Analysis identified loci affecting plant height, heading date and resistance Hessian fly, Fusarium head blight and to Septoria nodorum leaf and glume blotch. Accurate and repeatable methods for identifying genotypes with elite freezing tolerance are crucial to achieving the objectives of this research. Tolerance to over-wintering as well as unexpected spring-freeze events are crucial attributes of small grains if breeders are to improve the overall agronomics of winter cereals grown in the United States. Infrared thermal analysis and conventional histology used by ARS scientists at Raleigh, North Carolina in this project indicate that much of the existing literature regarding freezing processes in plants is inaccurate and may be one reason that progress developing freezing tolerance germplasm has been limited. In support of Objective 1D, barley and oat germplasm was evaluated by ARS scientists at Raleigh, North Carolina, at 9 and 13 locations respectively, worldwide. Thirty barley and 16 oat experimental lines were evaluated. Some locations reported no data due to flooding during spring and delivery difficulties. Techniques for investigating spring freeze in wheat were revised to account for differences in results obtained in field studies. Parents of a double haploid and several other genetically characterized populations are continuing to be evaluated. A detailed analysis of an unusual freezing pattern in wheat was completed by ARS scientists at Raleigh, North Carolina and was discovered using infrared thermography in 2017 with an international team of researchers. A manuscript was submitted and published. A revised 3D reconstruction technique with dye-infiltrated plants was developed that enabled rapid reconstruction of the vascular system in wheat, barley, oat and rye. A manuscript is in preparation. In support of Objective 2A, greenhouse seed was harvested by ARS scientists at Raleigh, North Carolina. Collaboration with scientists at North Carolina State University on the effect of freezing to preserve cut flowers was completed and a manuscript was submitted and published from 20 new RIL populations that were grown in the field at five locations in collaboration with regional breeding programs. Data were recorded for plant height, heading date, and level of epicuticular wax. Seed was harvested from approximately 5,000 head rows. Grain yield was recorded for RILs from four population grown in yield plots at five locations in North Carolina, South Carolina, Georgia and Virginia. In support of Objective 2B, germplasm in collaborative nurseries and breeding populations were genotyped by ARS scientists at Raleigh, North Carolina with markers associated with 59 different genes of interest to breeders. In support of Objective 2C, DNA sequence-based genotyping was done on more than 14,000 samples for trait mapping and genomic selection research. Sequence data from exome capture of 350 wheat lines of all market classes was analyzed and aligned the Chinese Spring reference genome to identify more than 800,000 polymorphisms. These data are being analyzed to identify signatures of selection in the different United States marker classes. Sequences flanking 2500 polymorphisms distributed across the wheat genome having high minor allele frequency in eastern germplasm were used to develop a target genotyping platform for genomic selection. Sequence data from this new technology are being analyzed. In support of Objective 3 during the 2020-2021 small grains growing season in North Carolina, ARS scientists grew 12 uniform trials from across the United States, as well as ARS Raleigh, North Carolina lines in Elite, Advanced, and Preliminary trials. In addition, ARS scientists grew 210 lines in first year yield trials, 3841 head-rows, and 189 segregating populations. These trials were distributed across five North Carolina locations (Kinston, Laurel Springs, Plymouth, Raleigh, and Salisbury). The Kinston, Laurel Springs, and Plymouth locations were lost due to poor stands. ARS scientists completed 239 new wheat crosses in the greenhouse, where we presumably combined multiple diseases resistances (stem rust, stripe rust, leaf rust, Fusarium head blight, powdery mildew, yellow dwarf virus, and glume blotch) with high grain yield and desirable agronomics. In barley, scientists completed 91 new crosses, looking to combine malting quality with winterhardiness, disease resistance, good grain yields, and desirable agronomics. Pythium root rot has caused significant losses in North Carolina wheat production due to severe stunting, and is likely to be an increasing problem as sea level rise and heavy rainfall events intensify. Little was known about the causal Pythium species or the conditions that favor this disease of wheat. ARS scientists have confirmed that the three most frequent species involved in root rot and stunting of wheat are Pythium irregulare, P. vanterpoolii, and P. spinosum. Further, in controlled environment experiments, disease was shown to be significantly more severe at 12/14°C compared to 18/20°C. A technique to screen wheat genotypes for tolerance or resistance to Pythium root attack has been developed and is being pilot-tested.

Accomplishments
1. Quantifying sensitivity of wheat powdery mildew to fungicides. ARS scientists at Raleigh, North Carolina, suggest the United States population of wheat powdery mildew had never been surveyed for sensitivity to either class of fungicides used to control it, quinone outside inhibitors (QoIs) or demethylation inhibitors (DMIs). In other countries, the wheat powdery mildew population has lost sensitivity to both modes of action. ARS researchers at Raleigh, North Carolina, conducted a survey of United States powdery mildew isolates from 15 central and eastern United States where wheat powdery mildew epidemics are common. Over 375 isolates were screened for sensitivity to two QoIs, pyraclostrobin and picoxystrobin, and two DMI fungicides, tebuconazole and prothioconazole. While none of the known mutations were detected that cause insensitivity to QoIs, a range of QoI sensitivity was observed, suggesting there is currently a quantitative erosion of efficacy. For DMIs, significant regional differences in sensitivity were observed with sensitivity lower in the eastern United States. The results will be publicized and used to encourage judicious use of fungicides. They show the importance of rotating between the two chemistry classes and reducing unnecessary applications in the United States in order to delay the loss of the only two fungicidal modes of action effective against wheat powdery mildew.

2. Characterizing genetics of plant growth informs genomic selection approaches in wheat . ARS scientists at Raleigh, North Carolina, believe genetic variation in growth over the course of the season is a major source of grain yield variation in wheat. While major genes underlying flowering time and plant height in wheat have been cloned, their importance in contributing to genetic variation for plant growth over time is not fully understood. ARS scientists utilized a population constructed from hybridization of two modern wheat cultivars adapted to the southeast United States to determine that almost all additive genetic variation in plant growth traits is associated with known major variants and novel moderate-effect quantitative trait loci (QTL). Major differences were observed in this population despite the similar plant height and heading date characters of the parental lines. This segregation is being driven primarily by a small number of mapped QTL, instead of by many small-effect, undetected QTL. As most breeding populations in the southeastern United States segregate for known genes for these traits, genetic variation in plant height and heading date in these populations likely emerges from similar combinations of major and moderate effect QTL. This information is being used to provide wheat breeders with more accurate and cost-effective genomic prediction models by targeted genotyping of key markers. This research will increase efficiency and reduce costs of genomics assisted development of improved wheat cultivars.

3. Identification of genes for resistance to Fusarium head blight (FHB) in eastern winter wheat. ARS scientists at Raleigh, North Carolina, suggest use of genetic resistance is one of the most important strategies to manage the devastating disease FHB in wheat. It is important to identify, characterize, and deploy sources of genetic resistance in adapted, elite lines so that new resistant cultivars do not suffer from poor agronomics. In collaboration with university researchers, ARS scientists identified quantitative trait loci (QTL) associated with FHB resistance in a high-yielding, moderately resistant soft red winter wheat cultivar ‘Jamestown’. Three populations constructed from hybridization of Jamestown with susceptible cultivars were evaluated for disease and characterized with DNA markers. Two new QTL, named QFHB.vt-1B.1 and QFHB.vt-1B.2, consistently contributed to FHB incidence, FHB severity, Fusarium-damaged kernels, and DON content. DNA marker assays were developed that are being widely used for marker-assisted selection of the QTL so that wheat breeding programs may develop FHB-resistant, high-yielding varieties.

4. Visualizing the vascular system of small grains in 3 dimensions. ARS scientists at Raleigh, North Carolina, suggest winter hardiness is one of the most important traits found in winter-sown small grains. To discover screening tools for identifying mechanisms responsible for winter hardiness it is important to visualize where freezing is initiated in plants. In small grains, freezing begins in the vascular system, specifically in water conducting vessels. ARS scientists previously developed a technique to visualize the vascular system of small grains in 3 dimensions using dye infiltration. Using this technique in wheat, barley, rye and oats, scientists discovered that a spherical compartment exists within the crown that likely is a reservoir of water that feeds individual leaves. A reconstruction of wheat that had been frozen indicated that specific cells at the periphery of the compartment were damaged. If there are differences between oat and wheat in the cells at the edge of the compartment this may be one explanation for the lack of winterhardiness in oat and why some oat lines are not as hardy as others. Information from this technique could provide a tool for breeders to select genotypes with elite freeze tolerant mechanisms and transfer those traits to agronomically acceptable genotypes so that winter oats can be grown further north within the United States.

5. Age dependent freezing in wheat leaves. ARS scientists at Raleigh, North Carolina, suggest one aspect of developing screening tools to discover winter hardy germplasm is to identify the reason certain tissues in the plant are more freezing tolerant than others. Infrared analysis of oats, wheat, and rye plants previously indicated that leaves freeze in an age-dependent sequence with older leaves freezing first; many times the youngest leaves never freeze and are more freezing tolerant than older leaves. In collaboration with an international group of scientists, ARS scientist at Raleigh, North Carolina analyzed the role of protein and carbohydrate concentrations, as well as the presence of bacterial and fungal populations and anatomical measurements. This new discovery helps explain why certain tissues in wheat never freeze and why a plant will survive winter even though much of the plant may be dead.

Review Publications
Carpenter, Wright, Malla, S., Singh, L., Van Sanford, D., Clark, A., Harrison, S., Murphy, J., Costa, J., Chao, S., Brown Guedira, G.L., Mcmaster, N., Schmale Iii, D.G., Griffey, C.A., Rawat, N. 2020. Identification and validation of Fusarium head blight resistance QTL in the U.S. soft red winter wheat cultivar ‘Jamestown’. Crop Science. 60:2919–2930. https://doi.org/10.1002/csc2.20307.
Megerssa, S.H., Ammar, K., Acevedo, M., Brown Guedira, G.L., Ward, B.P., Degete, A.G., Randhawa, M.S., Sorrells, M.E. 2020. Multiple-race stem rust resistance loci identified in durum wheat using genome-wide association mapping. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2020.598509.
Sallam, A.H., Manan, F., Bajgain, P., Martin, M., Szinyei, T., Conley, E., Brown Guedira, G.L., Muehlbauer, G.J., Anderson, J.A., Steffenson, B.J. 2020. Genetic architecture of agronomic and quality traits in a nested association mapping population of spring wheat. The Plant Genome. 13(3), e20051.
Guo, J., Khan, J., Pradhan, S., Shahi, D., Khan, N., Avci, M., Mcbreen, J., Harrison, S.A., Brown Guedira, G.L., Murphy, J.P., Johnson, J., Mergoum, M., Mason, R.E., Ibrahim, A.M., Sutton, R., Griffey, C., Babar, M.A. 2020. Multi-trait genomic prediction of yield-related traits in US soft wheat under variable water regimes. Genes. 11(11), 1270. https://doi.org/10.3390/genes11111270.
Hawkes, C.V., Kjoller, R., Raaijmakers, J.M., Riber, L., Christensen, S., Rasmussen, S., Christensen, J.H., Dahl, A.B., Westergaard, J.C., Nielsen, M., Brown Guedira, G.L., Hansen, L.H. 2021. Extension of plant phenotypes by the foliar microbiome. Annual Reviews of Plant Biology. 72:823-846. https://doi.org/10.1146/annurev-arplant-080620-114342.
Russell, B., Brown Guedira, G.L., Sneller, C.H., Mohammadi, M. 2020. Transferability of marker trait associations in wheat is disturbed mainly by genotype × year interaction. Crop Breeding, Genetics and Genomics. 2(3):e200013. https://doi.org/10.20900/cbgg20200013.
Ward, B.P., Merrill, K., Bulli, P., Pumphrey, M., Mason, R.E., Mergoum, M., Johnson, J., Sapkota, S., Brown Guedira, G.L. 2021. Analysis of the primary sources of quantitative, adult plant resistance to stripe rust in U.S. soft red winter wheat germplasm. The Plant Genome. 14:e20082. https://doi.org/10.1002/tpg2.20082.
De Witt, N., Guedira, M., Lauer, E., Murphy, J.P., Marshall, D.S., Mergoum, M., Johnson, J., Holland, J.B., Brown Guedira, G.L. 2021. Characterizing the oligogenic architecture of plant growth phenotypes informs genomic selection approaches in a common wheat population. BMC Genomics. 22:402. https://doi.org/10.1186/s12864-021-07574-6.
Hahn, E.A., Whetten, R.B., Cowger, C. 2021. Sensitivity of the U.S. Wheat Powdery Mildew Population to QoI Fungicides and Determination of the Complete Blumeria graminis f. sp. tritici cytochrome b gene. Phytopathology. https://doi.org/10.1094/PHYTO-04-21-0132-R.
Xue, A.G., Lim, S., Chen, Y., Humphreys, G., Cao, W., Menzies, J., Cowger, C., Li, H., Serajazari, M. 2021. Virulence structure of Blumeria graminis f. sp. tritici, the causal agent of wheat powdery mildew, in Ontario in 2018 and 2019. Canadian Journal of Plant Pathology. https://doi.org/10.1080/07060661.2021.1915876.
Reeves, E., Kerns, J., Cowger, C., Shew, B.B. 2021. Pythium spp. associated with stunting and root rot of winter wheat in North Carolina. Plant Disease. 105:986-996. https://doi.org/10.1094/PDIS-09-20-2022-RE.
Cowger, C. 2021. Key challenges in breeding durable disease-resistant cereals: North America. In Achieving Durable Disease Resistance in Cereals. R. Oliver, ed. Burleigh-Dodds Science Publishing Ltd., Cambridge, UK. Book Chapter. 978-1-78676-601-4.
Livingston, D.P., Bertrand, A., Wisniewski, M., Tisdale, R.H., Tuong, T.D., Gusta, L., Artlip, T.S. 2021. Factors contributing to ice nucleation and sequential freezing of leaves in wheat. Planta. 253:124. https://doi.org//10.1007/s00425-021-03637-w.
Livingston, D.P., Tuong, T.D. 2020. Using pixel-based microscope images to generate 3-D reconstructions of frozen and thawed plant tissue. Plant Cold Acclimation: Methods and Protocols. Springer Protocols, Second Edition. Book Chapter. pgs. 119-139.
Takahashi, D., Johnson, K., Tuong, T.D., Sampathkumar, A., Livingston, D.P., Kopka, J., Kuroha, T., Yokoyama, R., Nishitani, K., Zuther, E., Hincha, D. 2021. Cell wall modification by the xyloglucan endotransglycoslylase/hydroxylase XTH19 influences Arabidopsis thaliana freezing tolerance after cold and sub-zero acclimation. Plant Cell and Environment. 44:915-930.
Brown, J., Holloway, M., Dacosta, M., Bernstein, R., Lu, J., Tuong, T.D., Patton, A., Dunne, J., Arellano, C., Livingston, D.P., Milla-Lewis, S. 2021. Difference in proteome response to cold acclimation in Zoysia japonica cultivars with different levels of freeze tolerance. Crop Science. 60:2744-2756.
Brown, J., Yu, X., Holloway, M., Tuong, T.D., Schwartz, B., Patton, A., Arelano, C., Livingston, D.P., Milla-Lewis, S. 2021. Identification of QTL associated with cold acclimation and freezing tolerance in Zoysia japonica. Crop Science. https://doi.org/10.1002/csc2.20368.
Nogueira, M., Livingston, D.P., Tuong, T.D., Sinclair, T. 2020. Xylem vessel radii comparison between soybean genotypes differing in tolerance to drought. Journal of Crop Improvement. 34:404-413.
Pradhan, D., Bertin, D., Sinclair, T., Nogueria, M., Livingston, D.P., Carter, T. 2020. Microsphere stem blockage as a screen for nitrogen fixation drought tolerance in soybean. Physiologia Plantarum. https://doi.org/10.1111/ppl.13281.
Jahnke, N., Dole, J., Livingston, D.P., Bergman, B. 2019. Impacts of carbohydrate pulses and short-term sub-zero temperatures on vase life and quality of cut Paeonia lactiflora Pall. hybrids. Postharvest Biology and Technology. https://doi.org/10.1016/j.postharvbio.2019.111083.
Mashaheet, A., Burkey, K.O., Saitanis, C., Abdelrhim, A., Rafiullah, R., Marshall, D.S. 2020. Differential ozone responses identified among key rust susceptible wheat genotypes. Agronomy. 10:1853. https://doi.org/10.3390/agronomy10121853.