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

Location: Plant Science Research

2020 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
ARS researchers at Raleigh, North Carolina, evaluated 2,341 winter wheats from 37 U.S. public and private breeding programs in Kenya for resistance to stem rust and stripe rust. Approximately 564 of the wheats were from the ARS Raleigh, North Carolina location. Out of the 564 lines, 220 were resistant to stem rust. In addition, from the 220 stem rust resistant lines, 42 of them were also resistant to stripe rust. All of the stem and stripe rust resistant lines had major gene resistance combined with minor (or adult-plant) resistance genes. Data from our U.S. evaluations also showed that all of the 220 stem rust resistant lines also had resistance to leaf rust. ARS researchers at Raleigh, North Carolina, also evaluated 119 winter barleys from four U.S. public breeding programs in Kenya to stem and stripe rust. Nearly all of the 119 lines showed a moderate reaction to stem rust and a resistant reaction to stripe rust. In the 2019-2020 small grains growing season in North Carolina, ARS researchers at Raleigh, North Carolina, grew 12 Uniform trials from across the U.S., as well as lines in three Elite, five Advanced, and 2 Preliminary trials. In addition, ARS researchers at Raleigh, North Carolina, grew 530 lines in first year yield trials, 4500 head-rows, and 630 segregating populations. These trials were distributed across six North Carolina locations (Goldsboro, Kinston, Laurel Springs, Plymouth, Raleigh, and Salisbury). The Goldsboro (organic) location was lost to poor stands followed by flooding. About one-quarter of Laurel Springs was lost to poor stands and the remaining three-quarters were lost to freezing temperatures during heading and pollination in mid-May. The Kinston location had significant damage from Hessian-Fly, and harvest was preceded by 8 days of rains, which resulted in very high levels of preharvest sprouting. The Plymouth, North Carolina, Raleigh, North Carolina, and Salisbury, North Carolina, locations had minor levels of preharvest sprouting, but were otherwise moderately good. ARS researchers at Raleigh, North Carolina, completed 585 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, ARS researchers at Raleigh, North Carolina, completed 83 new crosses, looking to combine malting quality with winterhardiness, disease resistance, good grain yields, and desirable agronomics. ARS researchers at Raleigh, North Carolina, also completed our evaluation of selected wheats to ozone exposure, using continuous-stir reaction tanks and outdoor plant environment chambers. DNA sequence based genotyping was done by ARS researchers at Raleigh, North Carolina, on more than 10,000 samples for trait mapping and genomic selection. Exome capture and sequencing was performed on an additional 48 soft red winter wheat lines. These data were combined with sequences from 350 wheat lines of all market classes and analyses resulted in identification of polymorphisms used to construct a practical haplotype map graph of US wheat. High density genetic linkage maps were analyzed in conjunction with phenotypic data for soft red winter wheat mapping populations. Analysis identified loci affecting plant height, kernel size, leaf rust, stripe rust, powdery mildew resistance, as well as resistance to Septoria nordorum leaf and glume blotch. Germplasm in collaborative nurseries and breeding populations were genotyped with markers associated with 59 different genes of interest to breeders. New marker assays were developed for genes conferring resistance to powdery mildew, and Septoria nordorum glume blotch. Accurate and repeatable procedures for identifying genotypes with elite freezing tolerant mechanisms are crucial to accomplishing the objectives of this research. Both winter-freeze tolerance and tolerance to unexpected spring freeze events are crucial traits if breeders are to improve winter cereals grown in the United States. Infrared thermal analysis and conventional histology used in this project have determined that much of the existing literature regarding freezing processes in plants is incorrect and may be one reason why progress in developing freezing tolerance germplasm has been so slow. Twenty-eight barley and 20 oat experimental lines were evaluated by ARS researchers at Raleigh, North Carolina, at 9 and 13 locations respectively, worldwide. Many locations have no data to report from extensive flooding during spring. Data from last year indicated that 3 oat lines submitted by Prof Lapinski in Poland was significantly hardier than existing oat cultivars. 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 were evaluated. The international team of researchers organized in 2018, with the aim of understanding age-dependent freezing in small grains, continued a detailed analysis of freezing in wheat that was discovered using infrared thermography in 2017. Protein and carbohydrate concentrations as well as the presence of bacterial and fungal populations and anatomical measurements have all been collected and analyzed and a manuscript is being written. This research will help explain why certain tissues in wheat never freeze and how they contribute to the hardiness of wheat as compared to less freezing tolerant species such as barley and oat. A revised 3D reconstruction technique with dye-infiltrated plants was developed by ARS researchers at Raleigh, North Carolina, that enabled rapid reconstruction of the vascular system in wheat, barley, oat and rye. The technique revealed a tank-like structure within the crown of winter cereals where water is likely contained. The technique demonstrated that leaves probably draw water directly from this structure. The implications for freezing resistance or susceptibility are being investigated. Worldwide, wheat powdery mildew populations have shown a high capacity for adapting to become resistant to the two classes of fungicides that are used to manage them: demethylase inhibitors (DMIs) and quinone outside inhibitors (QoIs). Our lab has discovered that so far, the U.S. wheat powdery mildew population lacks the key known mutations that would render it highly resistant to DMIs or QoIs. However, ARS researchers at Raleigh, North Carolina, have revealed regional differences within the U.S. in sensitivity to DMIs, suggesting erosion of efficacy in some regions. The genetic basis for reduced DMI sensitivity has been investigated, and a mutation (Y136F) in the CYP51 gene that reduces sensitivity has been detected in some isolates. ARS researchers at Raleigh, North Carolina, have also determined there are differences in CYP51 expression and copy number that are associated with moderately reduced sensitivity. Together, these genetic and cellular differences create the potential for U.S. wheat powdery mildew to evolve to much higher levels of DMI insensitivity, as has happened in the UK and Europe. In collaboration with UK scientists, ARS researchers at Raleigh, North Carolina, are comparing U.S. and UK powdery mildew populations to gain new insight into the possible pathway to insensitivity that may lie ahead if fungicide chemistries are not properly rotated in the United States. ARS researchers at Raleigh, North Carolina, have also made novel discoveries concerning the powdery mildew mitochondrial gene mutations that underlie the differences in QoI sensitivity that we have observed. Together, these data are allowing us to understand the current status of evolution to fungicide resistance in the U.S. wheat powdery mildew population. This knowledge helps inform crop managers who are faced with decisions about fungicide selection and stewardship.

Accomplishments
1. New sources of wheat rust resistance. Stem, stripe, and leaf rust are global impediments to reliable wheat production. Genetic resistance is the most reliable and cost-effective form of rust resistance, particularly to the world’s poor. Scientists with ARS in Raleigh, North Carolina, have used traditional screening with the pathogens along with molecular marker technology and field evaluations in the U.S., east Africa, and Pakistan to develop sources of multiple rusts resistance. In the 2019-20 growing season, we confirmed from multiple field trials, that 42 lines developed by the researchers in Raleigh, North Carolina, had resistance to all three rusts. These lines are important to U.S. agriculture because they could be used directly as improved varieties or as germplasm for other breeders to use.

2. Identification of stripe rust resistance in soft red winter wheat. Stripe rust, or yellow rust, caused by the fungus Puccinia striiformis f.sp. tritici, has historically been a major yield-limiting disease in cooler wheat growing regions. However, starting in the year 2000, new strains of stripe rust adapted to warmer climates, putting Southern and Southeastern U.S. wheat growing regions at risk. ARS researchers in Raleigh, North Carolina, determined the genomic locations of different genes that play important roles in stripe rust resistance in the Southeastern growing region. They found that three locations on chromosomes 2A, 3B, and 4B acted as major resistance regions. The 3B and 4B regions had been previously reported by several researchers; the current study helped to determine how much they contribute to stripe rust resistance in the wheat lines typically used in regional breeding programs. They also determined that resistance due to a widely-used gene on 2A, Yr17, was likely overcome by the evolving stripe rust pathogen several years ago. However, this study supports the conclusions of ARS researchers in Washington state, who believe that a second resistance gene close to Yr17 offers continued resistance from the 2A region. Finally, this study found that there is room for further development of more disease resistant lines, as few of the lines tested combined resistance from all three of the major genomic regions.

3. Revealing fungal and mycotoxin diversity in Fusarium head blight. Fusarium head blight (FHB) is a globally damaging disease of small-grain cereals that causes major losses to U.S. growers and grain purchasers every year. Fusarium graminearum is the main fungal species that causes FHB in the U.S., and it contaminates grain with the mycotoxin deoxynivalenol (DON). In other countries, it has been shown that FHB is caused by a diversity of Fusarium species that produce mycotoxins other than DON, but very little was known about the possible occurrence of non-DON producers in U.S. small grains. A large-scale, intensive survey was undertaken by ARS scientists in Raleigh, North Carolina, across the state of North Carolina to explore the frequency and distribution of Fusarium species in scabby wheat heads. Wheat heads with FHB were sampled from 59 fields in 24 counties located in three distinct agronomic zones typical of the region east of the Appalachian Mountains. Surprisingly, although F. graminearum was the majority species detected, species in the Fusarium tricinctum species complex (FTSC) were frequent, and even dominant in some fields. These FTSC strains do not produce DON, but instead produce moniliformin and other “emerging mycotoxins.” This discovery is a first in the U.S., and has important implications for the study and management of FHB in this country. It raises the possibility that FHB-affected wheat and barley fields outside North Carolina may have similar, field-specific diversity of Fusarium species and mycotoxins. It shows the importance of sampling small-grain fields more intensively to detect “patches” of Fusaria producing moniliformin and other mycotoxins that have previously escaped detection.

4. Genotypic differences in freezing tolerance during reproductive phase of growth. Winter cereal crops in a reproductive phase of growth are much more susceptible to damage from unexpected spring freezes than they are in the fall during their vegetative stage of growth. A procedure developed in 2013 for evaluating wheat genotypes for spring-freeze damage was modified by ARS researchers at Raleigh, North Carolina, as a result of infrared observations during field evaluations. The procedure confirmed that contrary to existing literature there are genetic differences between wheat genotypes in spring freeze tolerance. Differences were found between parents of a double haploid population that has been genetically characterized, laying the groundwork for a detailed genetic characterization of spring freeze tolerance.

5. Basis for sequential freezing in wheat. Scientists investigating freezing tolerance have always wondered how certain plants can prevent freezing from occurring. Infrared analysis of oats, wheat, and rye plants during controlled freezing as well as natural conditions indicated that leaves freeze in sequence, with older leaves freezing first; many times the youngest leaves never freeze. A collaborative effort between ARS scientist at Raleigh, North Carolina, along with scientists world-wide demonstrated that this sequential freezing involves specific ice recrystalization proteins, carbohydrates, numerous populations of bacteria and fungi, as well as anatomical differences between leaves. Using these characteristics to screen germplasm for elite freezing tolerant mechanisms that could be incorporated into existing cultivars is an ongoing aspect of this research.

6. 3D reconstruction revealed a new vascular structure in wheat. Since winterhardiness primarily involves water freezing it is important to understand where high concentrations of water occur in plants since that is where most damage will occur. Infrared analysis of wheat demonstrated that freezing always begins in roots, where water concentration is highest and then proceeds upward through the vascular system. This was true even though leaves were always colder than roots. To better understand freezing patterns that initially follow the vascular system ARS scientist at Raleigh, North Carolina, developed a technique for visualizing water conducting vessels in plants that were infiltrated with dye. The technique revealed that winter cereals contain a tank-like structure inside the crown from which leaves draw water. Infrared analysis indicated that a major freeze event occurs in this region and there is a significant delay before freezing proceeds into leaves. Ongoing research will examine how this information can be used to identify freezing tolerant germplasm that can be crossed with existing cultivars and improve winterhardiness.

Review Publications
Lozada, D., Godoy, J.V., Murray, T.D., Ward, B.P., Carter, A.H. 2019. Genetic dissection of snow mold tolerance in US Pacific Northwest winter wheat through genome-wide association study and genomic selection. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2019.01337.
Lozada, D., Godoy, J.V., Ward, B.P., Carter, A.H. 2019. Genomic prediction and indirect selection for grain yield in US Pacific Northwest winter wheat using spectral reflectance indices from high-throughput phenotyping. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms21010165.
Cowger, C., Ward, B.P., Brown Guedira, G.L., Brown, J.K. 2020. Role of effector-sensitivity gene interactions and durability of quantitative resistance to septoria nodorum blotch in Eastern U.S. wheat. Frontiers in Plant Science. 11:155. https://doi.org/10.3389/fpls.2020.00155.
Cowger, C., Ward, T.J., Nilsson, K., Arellano, C., McCormick, S.P., Busman, M. 2020. Regional and field-specific differences in Fusarium species and mycotoxins associated with blighted North Carolina wheat. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2020.108594.
Mckee, G., Cowger, C., Dill-Macky, R., Friskop, A., Gautam, P., Ransom, J., Wilson, W. 2019. Disease management and estimated effects on DON (Deoxynivalenol) contamination in fusarium infested barleys. Open Access Journal of Agricultural Research. 9:155. https://doi.org/10.3390/agriculture9070155.
Cowger, C., Brown, J.K. 2019. Durability of quantitative resistance in crops: Greater than we know? Annual Review of Phytopathology. 57:253-277.
Meyers, E., Arellano, C., Cowger, C. 2019. Sensitivity of United States wheat powdery mildew (Blumeria graminis f.sp. tritici) populations to the demethylation inhibitor fungicides. Plant Disease. https://doi.org/10.1094/PDIS-04-19-0715-RE.
Cowger, C., Beccari, G., Dong, Y. 2020. Timing of susceptibility to Fusarium head blight in winter wheat. Plant Disease. https://doi.org/10.1094/PDIS-03-20-0527-RE.
Guttieri, M.J., Bowden, R.L., Reinhart, K., Marshall, D.S., Jin, Y., Seabourn, B.W. 2020. Registration of hard white winter wheat germplasms KS14U6380R5, KS16U6380R10, and KS16U6380R11 with adult plant resistance to stem rust. Journal of Plant Registrations. 1-7. https://doi.org/10.1002/plr2.20004.
Mehra, L., Adhikari, U., Ojiambo, P., Cowger, C. 2019. Septoria nodorum blotch of wheat. Plant Disease. https://doi.org/10.1094/PHI-I-2019-0514-01.
Cowger, C., Smith, J., Boos, D., Bradley, C.A., Ransom, J., Bergstrom, G.C. 2020. Managing a destructive, episodic crop disease: A national survey of wheat and barley growers’ experience with Fusarium head blight. Plant Disease. 104:634-648. https://doi.org/10.1094/PDIS-10-18-1803-SR.
Anderson, N.R., Freije, A.N., Bergstrom, G.C., Bradley, C.A., Cowger, C., Faske, T., Kleczewski, N., Padgett, G.B., Paul, P., Price, T., Wise, K.A. 2020. Sensitivity of Fusarium graminearum to metconazole and tebuconazole fungicides before and after widespread use in wheat in the United States. Plant Health Progress. 21:85-90. https://doi.org/10.1094/PHP-11-19-0083-RS.
Richards, J.K., Stukenbrock, E.H., Carpenter, J., Liu, Z., Cowger, C., Faris, J.D., Friesen, T.L. 2019. Local adaptation drives the diversification of effectors in the fungal wheat pathogen Parastagonospora nodorum in the United States. PLoS Genetics. 15(10):e1008223. https://doi.org/10.1371/journal.pgen.1008223.
Rehman, M.U., Gale, S.W., Brown-Guedira, G.L., Jin, Y., Marshall, D.S., Whitcher, L.C., Williamson, S.M., Rouse, M.N., Ahmad, J., Ahmad, G., Shah, I., Sial, M., Rauf, Y., Rattu, A., Ward, R.W., Nadeem, M., Ullah, G., Imtiaz, M. 2020. Identification of seedling resistance to stem rust in advanced wheat lines and varieties from Pakistan. Crop Science. 60:804–811. https://doi.org/10.1002/csc2.20056.
Mashaheet, A., Burkey, K.O., Marshall, D.S. 2019. Chromosome location contributing to ozone tolerance in wheat. Plants. 8:261.
Gaire, R., Huang, M., Sneller, C., Griffey, C., Brown Guedira, G.L., Mohammadi, M. 2019. Association analysis of baking and milling quality traits in an elite soft red winter wheat population. Crop Science. 59:1085–1094.
Kuzay, S., Xu, Y., Zhang, J., Katz, A., Pearce, S., Su, Z., Fraser, M., Anderson, J.A., Brown Guedira, G.L., Dewitt, N., Peters Haugrud, A., Faris, J.D., Akhunov, E., Bai, G., Dubcovsky, J. 2019. Identification of a candidate gene for a QTL for spikelet number per spike on wheat chromosome arm 7AL by high-resolution genetic mapping. Theoretical and Applied Genetics. https://doi.org/10.1007/s00122-019-03382-5.
Dewitt, N., Guedira, M., Lauer, E., Sarinelli, M., Tyagi, P., Fu, D., Hao, Q., Murphy, J. P., Marshall, D.S., Akhunova, A., Jordan, K., Akhunov, E., Brown Guedira, G.L. 2019. Sequence based mapping identifies a candidate transcription repressor underlying awn suppression at the B1 locus in wheat. New Phytologist. 225:326–339. https://doi.org/10.1111/nph.16152.
Daba, S.D., Tyagi, P., Brown Guedira, G.L., Mohammadi, M. 2019. Genome-wide association study in historical and contemporary U.S. winter wheats identifies height-reducing loci. The Crop Journal. 8:243-251. https://doi.org/10.1016/j.cj.2019.09.005.
Haaning, A.M., Smith, K.P., Brown Guedira, G.L., Chao, S., Tyagi, P., Muehlbauer, G.J. 2020. Natural genetic variation underlying tiller development in barley (Hordeum vulgare L). G3, Genes/Genomes/Genetics. Vol. 10, 4:1197-1212. https://doi.org/10.1534/g3.119.400612.
Brasier, K., Ward, B.P., Smith, J.H., Seago, J., Oakes, J., Balota, M., Davis, P., Fountain, M.O., Brown Guedira, G.L., Sneller, C., Thomason, W., Griffey, C. 2020. Identification of quantitative trait loci for nitrogen use efficiency in winter wheat. PLoS One. https://doi.org/10.1371/journal.pone.0228775.