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ARS Home » Pacific West Area » Corvallis, Oregon » Forage Seed and Cereal Research Unit » Research » Research Project #434578

Research Project: Development of Genetic, Genomic and Molecular Resources to Improve Performance, Adaptability and Utility of Cool Season Grasses and Cover Crops

Location: Forage Seed and Cereal Research Unit

2021 Annual Report

The long-term objective of this project is to improve the performance of grasses and cover crops. Specifically, during the next five years we will focus on the following objectives. Objective 1: Develop cover crops with increased performance and adaptability in end use environments. • Sub-objective 1A: Develop tools to select for acidic soil syndrome tolerant plants and breed tolerant annual ryegrass germplasm. (Hayes) • Sub-objective 1B: Improve annual ryegrass winter cover crop germplasm for reliable spring termination. (Hayes, Martin) Objective 2: Identify disease resistant germplasm in cool season grass species. • Sub-objective 2A: Evaluate grass cultivars (cvs) for susceptibility to Barley Yellow Dwarf Viruses. (Dombrowski, Martin) • Sub-objective 2B: Identify and evaluate choke resistant germplasm in orchardgrass. (Dombrowski, Martin) • Sub-objective 2C: Develop stem rust resistant germplasm and breeding tools in perennial ryegrass and determine the potential durability of resistance. (Hayes) Objective 3: Isolate endophytes from grasses found in arid regions to identify novel endophytes that improve persistence and performance of forage and turf related grasses in environments with limited water resources. Objective 4: Develop genetic and molecular resources that can be applied to reduce the impact of abiotic stresses on the adaptability and performance of grasses in diverse environments. • Sub-objective 4A: Sequence and annotate Lolium sp. genome for development of a public genome database. (Dombrowski, Martin) • Sub-objective 4B: Identify genes or pathways common to stress responses in multiple types of abiotic stress. (Dombrowski, Martin) • Sub-objective 4C: Evaluate Brachypodium overexpressing transcription factors for improved abiotic stress tolerance. (Dombrowski, Martin)

Forage, turf, and cover crop species are critical components of sustainable landscapes and agroecosystems. Most of the cool season grass seed in the United States is grown in the Pacific Northwest due to the mild winters and dry summers that are ideal for grass seed production. Development of adaptable, high-yielding, animal-compatible, low-input grass and cover crop cultivars are needed to enhance the utility of these crops in environments different from those of the Pacific Northwest, to expand their market potential, and meet the goals of improved food security. The challenges to the grass industry require a multifaceted research approach to develop genetic resources for improved adaptability and stress tolerance in grasses and cover crops to accelerate the pace of cultivar development. The research in this project will develop new selection techniques and breed germplasm of annual ryegrass with enhanced tolerance to acid soil syndrome and reliable spring termination when used as a cover crop (Objective 1). New grass germplasm, quantitative trait loci (QTL), and molecular markers linked to resistance QTL will be identified in order to reduce the impact of the diseases stem rust, choke and barley yellow dwarf virus on crop performance (Objective 2). The project will identify novel endophytes from grasses found in arid regions and test their ability to improve persistence and performance of forage and turf related grasses in environments with limited water resources (Objective 3). Transcriptome and whole genome sequencing along with gene function studies will develop the genetic and molecular resources needed to accelerate the breeding of new grass cultivars with improved performance. The development of biological, genetic, genomic and molecular resources from this project will lead to improved performance, adaptability and utility of cool season grasses and cover crops in diverse end use environments.

Progress Report
In support of Sub-objective 1A, a 96-well plate colorimetric assay for the ability of root exudates to chelate, or bind, aluminum was used to develop annual ryegrass populations improved for this trait. The ability of root exudates to chelate aluminum may improve the plant’s ability to tolerate aluminum toxic soils. The populations were developed by selecting 35 individuals with either high or low trait performance and allowing the selected plants in each group to intermate in isolation. A population using a group of randomly selected plants was developed as well. This procedure was repeated across four replicates, resulting in a total of 12 populations. Herbicide sensitivity in cover crops may improve the ease of their use by making cover crop termination and subsequent cash crop planting easier. Progress towards this goal was made as part of Sub-objective 1B by comparing populations of annual ryegrass selected for susceptibility to glyphosate to an unselected control population. Differences in glyphosate symptom severity between the two populations were small, but the selected population expressed more symptoms and died sooner after the herbicide application compared to the control population. Plants from the selected population also died when treated with lower glyphosate concentrations compared to the control population. Continued progress was made towards Sub-objective 2A on the evaluation of grasses for the presence of Barley yellow dwarf viruses and Cereal yellow dwarf virus (CYDV), the causal agents of barley yellow dwarf disease. Samples were collected for RNA-based barley yellow dwarf detection to confirm initial assay results. Progress was made toward Sub-objective 2B, the identification of choke resistant or tolerant orchardgrass germplasm. Currently, there is no way to control choke disease, which can cause up to 30% losses in orchardgrass seed yields in Oregon, where most of the U.S. orchardgrass seed is produced. Replicated field trials previously established to identify choke resistant or tolerant orchardgrass germplasm were maintained, and data was collected on flowering time and choke incidence from non-inoculated orchardgrass entries. Entries pre-inoculated with Epichloë typhina, the causal agent of choke, were evaluated for their ability to flower, potentially indicating their tolerance or ability to escape disease expression. Identification of choke resistant or tolerant germplasm offers a potential long-term solution for choke disease management in orchardgrass seed production fields. Stem rust caused by the fungus Puccinia graminis (pgl) is a destructive disease of perennial ryegrass grown for seed. Resistance, if effective against diverse isolates in diverse locations, could reduce the reliance on fungicide to control the disease. Field experiments were conducted to make progress on Sub-objective 2C. Field experiments with known resistant and susceptible perennial ryegrass clones were conducted at two sites in 2020 and 2021 to examine the stability of resistance. The experiments rely on natural infection by the local fungal strains. No disease occurred in 2020, and the 2021 tests are ongoing. More than 300 isolates of pgl have been collected from resistant and susceptible accessions for use in pgl diversity studies with simple sequence repeat (SSR) molecular markers. More than 170 previously published SSR markers were tested for the number of alleles produced from approximately eight isolates per SSR marker. Twenty-four SSR markers that produced more than three alleles were selected for further study, and software was used to design assay conditions for six multiplexed reactions of four SSRs per reaction. A population is being developed to identify molecular markers linked to genes that confer resistance to stem rust in perennial ryegrass. Seventeen clones of perennial ryegrass that capture broad diversity of the crop are being mated to a single clone with well-described resistance. Plants were vernalized over the winter and crossing is currently being conducted under greenhouse conditions. Project scientists continued progress towards developing molecular resources for perennial ryegrass as part of the National Turf Grass Sequencing Initiative to address Sub-objective 4A. Sequences from sensitive and drought-tolerant plants exposed to control and drought-treated conditions were annotated and are being analyzed to identify genes involved in drought tolerance. Additional experiments are currently in progress to further characterize drought-sensitive and drought-tolerant plants. Whole genome and transcriptome sequencing of a selected reference Lolium perenne cv Manhattan plant has been completed. In collaboration with scientists at Logan, Utah, the reference genome is being assembled and annotated. The development of publicly available molecular resources will facilitate research and crop breeding in perennial ryegrass. Drought and heat are two major stresses that often occur together and greatly affect crop production and yields. When exposed to stresses, plants need to sense the stresses, signal the presence of the stress throughout the plant, and elicit responses to alter plant growth to survive the stress. Continued progress was made towards Sub-objective 4B on identifying changes in gene expression when plants are exposed to a combination of drought and heat stress. We identified 20,221 unique differentially expressed sequences (DES) with increased expression and 17,034 unique DES with decreased expression within 48 hours of drought/heat exposure. These include DES encoding proteins involved in signaling, transporting molecules and proteins, regulating the expression of other genes, helping to fold proteins properly, making stress responsive hormones, and photosynthesis and respiration related processes. The identification of genes and pathways used by grasses to respond to drought and heat stress provide molecular resources that are necessary for developing new approaches to identify and develop grasses with greater tolerance to combined drought and heat stresses.

1. Analysis of choke expression in seedling-infected orchardgrass (Dactylis glomerata) germplasm. Choke is a serious disease in orchardgrass seed production fields in Oregon, where most U.S. orchardgrass seed is produced. ARS researchers in Corvallis, Oregon, showed that plants pre-inoculated with the choke-causing fungi at an early seedling stage gave rise to plants where all tillers were infected. After two years in the field, 76% of the pre-inoculated plants lacked flowers because the fungus “choked” the flower stalks, while plants that did flower also had choked flower stalks. Known choke-susceptible pre-inoculated cultivars had mainly choked tillers, while the putative resistant cultivars had more flowers and less choke. The use of pre-inoculated plants will be valuable for screening potential disease management strategies and for identifying germplasm with tolerance to choke, which are important for developing long-term solutions for controlling choke in orchardgrass seed production fields.

Review Publications
Merlet, L., Bushman, B.S., Dombrowski, J.E., Martin, R.C. 2021. Choke expression in Epichloë typhina seedling-infected orchardgrass (Dactylis glomerata) germplasm. Seed Production Research. 164:40-47.
Kucek, L.K., Azevedo, M.D., Eagen, S., Ehlke, N., Hayes, R.J., Mirsky, S.B., Reberg-Horton, C., Ryan, M.R., Wayman, S., Wiering, N.P., Riday, H. 2021. Seed dormancy regulated by genotype and environment in Hairy vetch (Vicia villosa Roth). Agronomy Journal. 10(11). Article 1804.
Bushman, B.S., Robbins, M.D., Warnke, S.E., Martin, R.C., Harris-Shultz, K.R., Amundsen, K.E. 2020. Gene expression differences for drought stress response in cool-season turfgrasses. International Turfgrass Society Research Journal.
Amundsen, K., Warnke, S.E., Bushman, B.S., Robbins, M.D., Martin, R.C., Harris-Shultz, K.R. 2020. Colonial bentgrass transcripts-expression differences compared with creeping bentgrass in response to water-deficit stress. 61(3):2135-2147. Crop Science.
Ahn, S., McDonnell, R., Corcoran, J., Martin, R.C., Choi, M.Y. 2020. Identification and functional characterization of the first molluscan neuromedin U receptor in the slug, Deroceras reticulatum. Scientific Reports. 10. Article 22308.
Kesoju, S., Kramer, M.H., Brunet, J., Greene, S.L., Jordan, A., Martin, R.C. 2021. Gene flow in commercial alfalfa (Medicago sativa subsp. sativa L.) seed production fields: Distance is the primary but not the sole influence on adventitious presence. PLoS ONE. 16(3). Article e0248746.