Location: Forage Seed and Cereal Research Unit
2024 Annual Report
Objectives
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)
Approach
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
This is the final report for project 2072-21000-054-000D, which has been replaced by new project 2072-21500-001-000D, titled, “Development of Plant and Molecular Resources to Improve Performance, Utility, and Tolerance of Cool Season Grasses and Legumes”.
Crop production practices may result in soil acidification, which can reduce crop growth by limiting essential plant nutrients and increasing toxic cations, particularly aluminum (Al3+). Chemical compounds that chelate Al3+ are found in plant root exudates and are known to condition tolerance to Al3+ toxicity. ARS scientists in Corvallis, Oregon, developed a high throughput method to measure root exudate chelation capacity of individual grass seedlings as part of research to address Sub-objective 1A. A selection experiment in annual ryegrass using this assay showed the data from the colorimetric assay is heritable trait that can be selected for in breeding populations.
Herbicide sensitivity in cover crops may improve the ease of cover crop termination and make subsequent cash crop planting easier. Progress towards this goal was made as part of Sub-objective 1B. In vitro leaf assays using a large range of glyphosate concentrations were unable to discriminate between herbicide susceptible and tolerant annual ryegrass plants. Tests using glyphosate applied to greenhouse-grown plants identified a sub-lethal dose that elicited symptoms but did not kill plants or prevent reproduction. The greenhouse assay was used to select populations of annual ryegrass with susceptibility to glyphosate and to compare the population 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.
Research to advance Barley yellow dwarf viruses (BYDVs) and Cereal yellow dwarf virus (CYDV) control addresses Sub-objective 2A. Field experiments with 25 varieties each of tall fescue, fine fescue, perennial ryegrass and orchardgrass were used to assess resistance or tolerance to BYDVs and CYDV. Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine virus infection and for strain identification. Fine fescues were the least infected, followed by perennial ryegrass and orchardgrass, with tall fescues being the most infected. BYDV-PAV was the most prevalent virus found in tall and fine fescues and perennial ryegrass, while CYDV was the most prevalent virus found in orchardgrass.
Research was conducted on stem rust of perennial ryegrass grown for seed as part of Sub-objective 2C. Stability of stem rust resistance was tested in more than 30 clones varying for resistance in ten multi-location field experiments. Resistant clones consistently expressed low disease. Based on these results, a population was developed from 18 clones to identify molecular markers linked to genes that confer resistance to stem rust in perennial ryegrass. The diversity of the stem rust fungal pathogen, Puccinia graminis f. sp. lolii (Pgl), is unknown. Simple sequence repeat (SSR) markers were used to assess the diversity in isolates collected from seed producing areas. Twenty-one SSR markers were used to genotype Pgl isolates collected from six field sites spread from the North to the South Willamette Valley of Oregon. Sample sizes from each site ranged from five to 48 isolates. The markers generated three to seven alleles each, resulting in 32 multi-locus genotypes. Most isolates were closely related to each other, with a few genetically distinct isolates. Pgl samples from a common site did not cluster together on a dendogram. These results demonstrate that the Willamette Valley population of Pgl from perennial ryegrass has genetic variation. Most of the genetic variation can be found within a collection site and there is no evidence of unique subpopulations of Pgl occupying specific Willamette Valley locations.
About 95% of U.S. orchardgrass seed is produced in Willamette Valley, Oregon, and is afflicted by the disease choke, caused by the fungus Epichloë typhina. The pathogen is present in almost every orchardgrass seed production field, which leads to yield losses as great as 30%. At present the best long-term solution to disease management for choke is host resistance, which is addressed in Sub-objective 2B. Research identified choke resistant plants and parents for breeding resistant orchardgrass. Progeny from the resistant plants were separated into maturity groups, evaluated for resistance to choke and interbred to develop plant material for eventual release.
Tissue culture is one of the first necessary steps to perform Agrobacterium-mediated transformation and gene editing. These tools together will be used to understand gene function and improve crop traits via molecular methods. In support of Objective 4, tissue culture research developed protocols for the model grass Brachypodium distachyon and crop species Lolium multiflorum (annual ryegrass) that successfully converted plant embryos into undifferentiated callus and back to differentiated plant tissues.
With diminishing water resources, development of drought tolerant grass varieties with reduced inputs is essential. Research that advances Sub-objectives 4A and 4B seeks to better understand the genetics of drought tolerance in grasses. Four endophyte free Lolium perenne grass lines (the cultivar Manhattan, and two drought sensitive and one drought tolerant line) were grown under controlled drought and well- watered conditions to study gene expression under drought conditions. As part of this work, 73 initial late embryogenesis abundant (LEA) protein-encoding genes were identified within the perennial ryegrass cv Manhattan genome. These genes have been associated with abiotic stress response in many other species.
For Sub-objectives 4A and 4B, ARS researchers are developing molecular resources for perennial ryegrass as part of the National Turf Grass Sequencing Initiative. In collaboration with ARS scientists at Logan, Utah, an individual Lolium perenne cv Manhattan plant has been selected for whole genome sequencing, and the genome is nearing publication. Flowering stalk, stressed root and leaf, and control tissue have been collected from this plant for RNA-sequencing with the objective of developing a more complete transcriptome for perennial ryegrass. The genomic diversity within annual ryegrass is being studied using whole genome sequencing of four samples from each of three annual and three biennial varieties. The goal is to identify the genomic variation that distinguishes these two types of annual ryegrass, focusing on single nucleotide polymorphisms (SNPs), small insertions or deletions (indels), and large-scale structural variation. This work is valuable for understanding the variation in flowering time within this species.
Progress for Sub-objective 4B was made in the analysis of genes or pathways involved in abiotic stress responses (Sub-objective 4B) using transcriptome (all genes expressed in an organism) analysis of genes induced or repressed in Lolium temulentum (Lt) in response to wounding. ARS researchers identified differentially expressed genes or sequences (DES), 9413 unique DES with increased expression and 7,704 unique DES with decreased expression within 24 hours of wounding. Analysis of these sequences revealed genes coding for proteins involved in signaling, stimulus and stress response, sequences involved in the biosynthesis of stress hormones, and proteins for growth and stress related pathways. These resources from the identified gene sequences will be used to develop approaches that can improve the recovery, regrowth and long-term fitness of forage and turf grasses before/after cutting or grazing. Drought and heat are two major stresses that often occur together and affect crop production and yields. Research identified changes in gene expression when plants are exposed to a combination of drought and heat stress. ARS researchers identified 20,221 unique 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 expression of other genes, helping fold proteins properly, making stress responsive hormones, and in photosynthesis and respiration processes. The identification of genes and pathways used by grasses to respond to drought and heat stress provide molecular resources for developing new approaches to identify and develop grasses with greater tolerance to combined drought and heat stresses.
Progress was made on Sub-objective 4C, by evaluating Brachypodium overexpressing specific transcription factors (TF), regulatory proteins that modulate expression of other genes, for improved stress tolerance. Research determined that plants overexpressing a selected TF (TF-OE) were shorter than wild type Brachypodium (WT), but this difference was less pronounced in plants grown under salinity stress. Analysis of DES between WT and TF-OE plants revealed many of the DES were like those often observed in response to abiotic and biotic stress encoding signaling proteins, stress and disease responsive proteins, oxidases/reductases, transcription factors, and enzymes/proteins involved in hormone production and function. Many of the DES between the WT and TF-OE under control conditions were also differentially expressed in WT in response to salinity stress. This suggests that this transcription factor is integral in abiotic stress responses, but that overexpressing this protein is placing the plant in a constant state of stress response, which may contribute to the plants reduced stature.
Accomplishments
1. Orchardgrass germplasm with resistance to choke. Choke is a prolific pathogen that reduces the seed yield in established orchardgrass in both seed and pasture fields. The knowledge around the transmission of choke is not well understood and any insights into how the pathogen spreads will aid producers in controlling the disease and managing its spread. Release of choke resistant germplasm by the ARS researchers in Corvallis, Oregon, will assist breeders in the development of resistant varieties that will benefit end-users.
