Location: Plant Science Research
2023 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
This is the final report for the project 6070-22000-018-000D which terminated in March 2023. In support of Objective 1, 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, heading date and resistance leaf rust, stripe rust, powdery mildew, Hessian fly, Fusarium head blight (FHB) and Septoria nodorum leaf and glume blotch. Data from screening nurseries in Objective 1 were used for genomic selection efforts in Objective 2.
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 (US). Infrared thermal analysis and conventional histology used 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 at 9 and 13 locations, respectively, worldwide until 2020 when disruptions in global nursery evaluation occurred due to Covid-19. Thirty barley and 16 oat experimental lines were evaluated each year. 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.
A detailed analysis of an unusual freezing pattern in wheat was completed that 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 Objectives 1 and 2, greenhouse seed was harvested from 20 new recombinant inbred lines (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 over three growing seasons. 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, each year germplasm in collaborative nurseries and breeding populations were genotyped with markers associated with 81 different genes of interest to breeders. Work was done to develop new Kompetittive Alelle Specific PCR assays associated with genes for resistance to powdery mildew, cereal rusts, FHB, and Hessian fly.
In support of Objective 2C, each year DNA sequence-based genotyping was done on more than 15,000 wheat samples and 1,200 barley samples for trait mapping and genomic selection research. In total, sequence data from exome capture of 192 eastern wheat lines of all market classes was analyzed and aligned the Chinese Spring reference genome to identify more than 800,000 polymorphisms. These data were analyzed in conjunction with ~450 wheat lines from all market classes to identify signatures of selection in the different US marker classes. Sequences flanking 2,500 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 were analyzed and provided to collaborators.
In support of Objective 3, each year, we grew 12 Uniform trials from across the U.S., as well as ARS-Raleigh, North Carolina, lines in Elite, Advanced, and Preliminary wheat and barley trials. In addition, each year we grew approximately 200 lines in first year yield trials, 5,000 head-rows, and 200 segregating populations. These trials were distributed across five North Carolina locations (Kinston, Laurel Springs, Plymouth, Raleigh, and Salisbury). We completed an average of 250 new wheat crosses in the greenhouse, where we presumably combined multiple diseases resistances (stem rust, stripe rust, leaf rust, FHB, powdery mildew, yellow dwarf virus, and glume blotch) with high grain yield and desirable agronomics. In barley, we completed on average 100 new crosses, looking to combine malting quality with winter hardiness, 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 rises and heavy rainfall events intensify. Little was known about the causal Pythium species or the conditions that favor this disease of wheat. In team research, we 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.
In support of Objective 4, a highly novel discovery was made concerning wheat powdery mildew resistance gene Pm1a. This gene provides resistance to Blumeria graminis f. sp. tritici (Bgt), the fungus that causes wheat powdery mildew. Pm1a has had greater-than-expected durability in the US, and when virulence to it has appeared, it has been surprisingly short-lived. A study of virulence to Pm1a was conducted using 216 Bgt isolates from six countries. It revealed that a single previously identified effector gene (AvrPm1a) on Bgt chromosome 6 was insufficient by itself to explain global patterns of virulence and avirulence to Pm1a. A genome-wide association study revealed a second effector locus on a different Bgt chromosome that also interacts with Pm1a. This highly unusual discovery was confirmed by a co-expression experiment, and the results were published in New Phytologist. The two-effector model for virulence to Pm1a is likely at least a part of the explanation for the greater durability of Pm1a in the U.S.
Fusarium head blight, also known as scab, is a fungal disease that attacks small grains, contaminating the grain with the mycotoxin deoxynivalenol (DON), a toxic compound also known as vomitoxin. For barley, the most common grain used to make malt for beer and spirits, even a small amount of DON can cause crops to be rejected by purchasers. In a four-year study, the Raleigh, North Carolina, Plant Science Research Unit team assessed three different fungicides for FHB reduction. They also evaluated the amount of DON in mature winter barley heads following a fungicide application at one of three growth stages – half heading, full heading, and six days after full barley head emergence. The latest fungicide timing reduced DON significantly more than the early timing for all three fungicides tested. Applying fungicide before all heads were emerged did not significantly reduce DON in winter barley as compared to not spraying at all. If scab is threatening, growers should wait about six days after barley heads have all appeared before applying fungicide. The results were published in Plant Disease and ARS put out a media release.
Accomplishments
1. Determining best practices to manage Fusarium head blight in winter barley. Winter barley is a crop that commands growing interest in the eastern United States (US) for malting potential, but malt barley purchasers have extremely low tolerance for the mycotoxin deoxynivalenol (DON) that is produced during Fusarium head blight (FHB) epidemics. ARS researchers in Raleigh, North Carolina, have established that to minimize DON in winter barley, growers should both plant moderately resistant varieties and (if there is FHB risk) apply a recommended DMI (triazole) fungicide. However, research-based findings were lacking when it comes to the optimal timing for FHB-targeted fungicide application to barley, particularly for winter barley. Fungicide manufacturers and spring barley workers were recommending timings of 50% and 100% head emergence that were not apparently based on replicated research, and that was confusing to producers. The ARS researchers’ 4-year replicated field study demonstrated that a later timing (6 days after 100% head emergence) is superior for minimizing FHB and DON in winter barley. The study also showed that 50% head emergence was equivalent to no fungicide at all when it came to DON. The results are important to producers of winter barley throughout the eastern U.S., and to purchasers of the crop such as maltsters and brewers. They will save millions of dollars by increasing the acceptability of barley produced during FHB epidemic years. The results are being shared widely through the US Wheat & Barley Scab Initiative and a media release from ARS.
2. Unusual discovery in wheat resistance to powdery mildew provides insight into durability. A highly novel discovery was made concerning wheat powdery mildew resistance gene Pm1a. This gene provides resistance to Blumeria graminis f. sp. tritici (Bgt), the fungus that causes wheat powdery mildew. Pm1a has had greater-than-expected durability in the United States (US), and when virulence to it has appeared, it has been surprisingly short-lived. ARS researchers in Raleigh, North Carolina, conducted a study of virulence to Pm1a using 216 Bgt isolates from six countries. It revealed that a single previously identified effector gene (AvrPm1a) on Bgt chromosome 6 that was known to interact with Pm1a was nevertheless insufficient by itself to explain global patterns of virulence and avirulence to Pm1a. A genome-wide association study revealed a second effector locus on Bgt chromosome 8 that also interacts with Pm1a. This highly unusual “two-gene” discovery was confirmed by a co-expression experiment, and the results were published in New Phytologist. The two-effector basis for virulence to Pm1a is likely at least a part of the explanation for the greater durability of Pm1a in the U.S., as separate mutations in each effector are needed to allow the fungus to infect wheat that carries Pm1a. The discovery is of strong interest to the scientific community researching host-pathogen interactions, and suggests that wheat breeders looking for durable resistance should use Pm1a in geographic regions where virulence is not yet elevated.
3. Awns improve yield when wheat experiences heat stress during grain fill. In an era of big data, scientists can leverage historical data sets to evaluate the utility of alleles across climate scenarios and inform selection of climate adapted varieties. Consistent yearly collaboration between USDA-ARS at Raleigh, North Carolina, and collaborators has generated large datasets consisting of genotypes and phenotypes of diverse germplasm across many locations and years. The scale of this historic data, particularly the large number of trials with distinct location x year environments across the soft wheat growing region that extends from Florida and South Texas to New York and Michigan, invites investigation of the relationship between environmental conditions and altered grain yield. The presence or absence of awns – whether a wheat line is ”bearded” or ”smooth” – is the most visible trait distinguishing wheat cultivars. Previous studies by our group identified the gene underlying awn suppression in soft winter wheat germplasm and identified a diagnostic marker for this trait. ARS scientists at Raleigh have used genotypic information about presence or absence of awns in wheat breeding lines in combination with grain yield and climate data to estimate the yield effects of awns under different environmental conditions over a 12-year period in the southeastern United States (US). We found that in some environments, absence of awns was associated with higher yields, but presence of awns was associated with better performance in heat-stressed environments more common at southern US locations. This approach provides breeders information on developing varieties with improved abiotic stress tolerance in their individual target environments. Wheat breeders in environments where awns are only beneficial in some years may consider selection for awned lines to reduce year-to-year yield variability, and with an eye towards future climates.
4. A public resource model for using genomic selection to improve scab resistance in wheat. Collaborative research between the ARS at Raleigh, North Carolina, and public breeding programs is aimed at efficient development of disease resistant, high-yielding wheat varieties for United States growers. Fusarium head blight, or head scab, is an economically and environmentally concerning disease of wheat since the pathogen produces a mycotoxin detrimental to human health. Efforts by plant breeders to develop resistant cultivars are complicated by the environmental effects on disease level and complex inheritance of resistance. We designed a study utilizing a genomic prediction approach for efficiently selecting the best lines. We combined data from collaborative field evaluation of advanced wheat lines developed by public breeding programs from 2010 to 2021 for which data on thousands of DNA variants was also generated. Given the environmental effects on disease development and the large number of years by location combinations in our dataset, we wanted to evaluate the value of retaining only data from environments having highly useful information. We determined that careful curation of the disease data based on a set of common check varieties was critical for producing high forward prediction accuracies for the traits percent Fusarium damaged kernels and content of the mycotoxin deoxynivalenol. This work provides a model for utilizing public resources for genomic prediction of resistance traits across wheat breeding programs.
Review Publications
Kloppe, T., Boshoff, W., Pretorius, Z., Lesch, D., Akin, B., Morgounov, A., Shamanin, V., Kuhnem, P., Murphy, P., Cowger, C. 2022. Virulence of Blumeria graminis f.sp. tritici in Brazil, South Africa, Turkey, Russia and Australia. Frontiers in Plant Science. 10:3389. https://doi.org/10.3389/fpls.2022.954958.
Kloppe, T., Whetten, R.B., Kim, S., Powell, O., Luck, S., Douchkov, D., Whetten, R., Hulse-Kemp, A.M., Balint Kurti, P.J., Cowger, C. 2023. Two pathogen loci determine Blumeria graminis f. sp. tritici virulence to wheat resistance gene Pm1a. New Phytologist. 238:1546-1561. https://doi.org/10.1111/nph.18809.
Van Sanford, D., Clark, A.J., Bradley, C.A., Brown Guedira, G.L., Cowger, C., Dong, Y., Baik, B.V. 2023. Registration of ‘Pembroke 2021’ soft red winter wheat. Journal of Plant Registrations. 17:376-384. https://doi.org/10.1002/plr2.20271.
Adhikari, U., Cowger, C., Ojiambo, P. 2023. Evaluation of a model for predicting onset of Septoria nodorum blotch in winter wheat. Plant Disease. 107:1122-1130. https://doi.org/10.1094/PDIS-06-22-1469-RE.
Liu, L., Griffey, C., Brooks, W., Seago, J., Fitzgerald, J., Thomason, W., Hokanson, E., Behl, H., Oakes, J., Pitman, R., Dunaway, D., Vaughn, M., Lee, M., Barnett, M., Custis, J., Chen, R., Fountain, M., Marshall, D., Cowger, C., Cambron, S., Jin, Y., Chen, X., Beahm, B., Browning, P., Hardiman, T., Osborne, S., Lin, C., Mennel, D., Santantonio, N. 2022. Registration of ‘Hardy 2519’ wheat. Journal of Plant Registrations. 16:385-393. https://doi.org/10.1002/plr2.20221.
Langridge, P., Alaux, M., Almeida, N., Ammar, K., Baum, M., Bekkaoui, F., Bentley, A., Beres, B., Berger, B., Braun, H., Brown Guedira, G.L., Burt, C.E., Caccamo, M., Cattivelli, L., Charmet, G., Civan, P., Cloutier, S., Cohan, J., Devaux, P., Doohan, F., Dreccer, F., Ferrahi, M., German, S., Goodwin, S., Griffiths, S., Guzman, C., Handa, H., Hawkesford, M.J., He, Z., Huttner, E., Ikeda, T.M., Kilian, B., King, I.P., King, J., Kirkegaard, J.A., Lage, J., Le Gouis, J., Mondal, S., Mullins, E., Ordon, F., Ortiz-Monasterio, J.I., Ozkan, H., Ozturk, I., Pereyra, S.A., Pozniak, C.J., Quesneville, H., Quincke, M.C., Rebetzke, G.J., Reif, J.C., Saavedra-Bravo, T., Schurr, U., Sharma, S., Singh, S.K., Singh, R.P., Snape, J.W., Tadesse, W., Tsujimoto, H., Tuberosa, R., Willis, T.G., Zhang, X. 2022. Meeting the challenges facing wheat production: The strategic research agenda of the global wheat initiative . Agronomy Journal. 12(11):2767. https://doi.org/10.3390/agronomy12112767.
Winn, Z.J., Larkin, D.L., Lozada, D.N., Dewitt, N., Brown Guedira, G.L., Mason, R. 2023. Multivariate genomic selection models improve prediction accuracy of agronomic traits in soft red winter wheat. Crop Science. 63(4):2115-2130. https://doi.org/10.1002/csc2.20994.
