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

2022 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 1a, our study of Septoria nodorum blotch (SNB), caused by the fungal pathogen Parastagonospora nodorum on wheat, has helped to identify an important wheat trait of resistance: slower lesion growth. This trait was shown to vary widely between susceptible and moderately resistant wheat genotypes. Further, while it was already known that relative humidity and temperature influence SNB development, our study showed that lesion growth in the susceptible genotypes are more strongly affected by increases in relative humidity and temperature in the susceptible genotypes than in the resistant ones. This indicates that an aspect of the susceptibility trait is environmental sensitivity. A paper on these findings is submitted and under revision. In support of Objective 2a, a set of heterozygous inbred lines segregating for an adult plant resistance gene on chromosome 3BS was tested for reaction for leaf rust in the field. Similar projects to characterize resistance to stem rust and powdery mildew in cultivar AGS2000 are ongoing. In support of Objective 2b, DNA was isolated for wheat lines entered into the eastern regional uniform nursery program and other collaborative testing nurseries as well as breeder submitted samples, for a total of 1536 samples. Lines were evaluated with a suite of markers related to genes for plant growth and development, quality and disease and insect resistance. Genotypic data were reported to collaborators. In support of Objective 2c, the Allegro genotyping platform that utilizes single primer extension technology (SPET) is being evaluated. Advantages of the platform include lack of up-front cost for development of an array of probes and no need to commit to large numbers of samples for any single design. A pool of primers for the Allegro targeted genotyping platform was developed for single nucleotide polymorphisms (SNP) selected from exome capture data of 400 North American wheat cultivars and breeding lines. Weighted linkage disequilibrium thinning was used to select targets based on minor allele frequency across all growing regions and within the Eastern United States winter wheat germplasm as well markers to fill gaps in genome-coverage. Our preliminary analysis using this technology is very encouraging, but it highlighted the importance of selecting high-quality SNP for inclusion in future primer pool designs. Multiple analyses are ongoing, including genotyping of causal variants (that are often insertions or deletions) included in the probe pools. Overall, our preliminary analyses suggest that the Allegro protocol can be used to capture thousands of loci, including markers targeting genes and loci of importance, with high accuracy and very little missing data. Cross-validation in the eastern winter wheat training population for resistance to Fusarium head blight is underway to access utility of markers in the Allegro mid-density panel for genomic prediction in this germplasm. We have gone through a second round of primer pool design that is being tested. Accurate and repeatable methods for identifying genotypes with elite freezing tolerance are crucial to meeting objective 1d research goals. 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. Because heat is given off when water freezes and infrared cameras can detect heat, recordings of plants with infrared cameras while they freeze can confirm the temperature at which freezing occurs and how ice spreads throughout the plant. In this project, infrared thermal analysis with high resolution infrared cameras in combination with conventional histology demonstrated 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. Techniques for investigating spring freeze in wheat in our lab were revised to account for differences in results obtained in field studies. A freezing tolerant oat line was identified and is being evaluated for a germplasm release. 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 from this research was published. In support of Objective 4a, results were analyzed on a global study of powdery mildew isolates’ virulence to wheat resistance (Pm) genes. The study compared virulence to long-available, low-numbered Pm genes with virulence to new Pm genes introgressed during the last two decades by a North Carolina State Univeristy Small Grains Breeder into wheat germplasm adapted to the United States Southeast region. A newly accepted manuscript provides details on which of the newer Pm genes are effective in which global regions where wheat powdery mildew is an emerging issue, including South Africa, Australia, and Egypt. A study of virulence to one wheat gene that confers resistance to powdery mildew, Pm1a, revealed that a single previously identified pathogen effector gene (AvrPm1a) 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 chromosome of the same pathogen, Blumeria graminis f. sp. tritici, that apparently also interacts with Pm1a. This is a novel discovery and co-expression experiments to confirm it are underway.

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 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. Research by an ARS scientist at Raleigh, North Carolina, has established that to minimize DON in winter barley, growers should both plant moderately resistant varieties and (if there is FHB risk) apply a recommended triazole fungicide. Research-based findings have been lacking when it comes to the optimal timing for FHB-targeted fungicide application to barley, particularly for winter barley. The recommendation to spring barley growers in the upper Plains is to apply fungicide when barley spikes have just emerged from the boot. However, our results indicate that a later timing (6 days after 100% spike emergence) is superior for minimizing FHB and DON in winter barley. This information is being shared with the barley community and will positively impact winter barley producers looking to reducing FHB and DON in the crop.

2. A novel approach to understanding wheat yield variation across environments. Plant breeders are interested in developing high yielding varieties with stable performance. However, grain yield is a genetically complex trait that varies across environments. In wheat, one approach to understanding the genetics of yield is to investigate its component traits; for example, grain number per plant or grain size. However, it is difficult to understand the relationships between genes that alter each component as increases in some traits may lead to compensatory decreases in other traits. Using molecular marker data, ARS scientists in Raleigh, North Carolina, proposed and implemented a unified modeling framework that first identifies genes affecting plant development and yield components, then allows investigation of the effects of these loci all at once. They demonstrated that the importance of a gene’s effect on grain yield results from a combination of effects on different yield components and on the different relationships between these components in each environment. Modeling the complicated effects of individual genes on many traits will allow wheat breeders to better understand which genes are useful in improving yield in a target environment. These results can inform strategies to better make predictions of yield potential for unobserved varieties using molecular marker data (301/3/3b).

3. High-yielding, disease resistant variety Catawba. Most of the wheat grown in the eastern U.S. is soft red winter wheat and most of the breeding effort is directed toward this market class. However, for millers and bakers in the eastern United States to meet the diversity of flour and subsequent product demands of their customers and consumers, hard wheat grain is needed that must be transported long distances. Locally adapted hard wheats could benefit the entire wheat production supply chain in the eastern United States and may reduce the environmental impact of long-distance grain transport. ARS scientists at Raleigh, North Carolina, have released a hard red winter wheat variety ‘Catawba’ with parentage from both soft red winter wheat varieties from the eastern US and hard red winter wheat varieties from the Great Plains. This variety has moderate resistance to Fusarium head blight (FHB) that is a challenge for cultivating hard wheat in the eastern United States, as accumulation of the mycotoxin deoxynivalenol (DON) in grain from FHB-infected plants has detrimental health effects and precludes milling of grain. Catawba has higher yields than previous releases and the robust resistance to FHB is likely generated by a combination of novel genes. Cultivation of ‘Catawba’ in the eastern United States may help supplement hard winter wheat from the Great Plains, especially in years where supply chain constraints and drought conditions place a high price on shipped grain.

U.S. DEPARTMENT OF AGRICULTURE

Plant Science Research: Raleigh, NC