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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

2020 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
Roots of annual ryegrass exude chemical compounds, and some of these may chelate heavy metals such as aluminum. The ability or capacity of root exudates to chelate aluminum and other heavy metals may condition tolerance to acidic soils. A high-throughput color change assay for root exudate chelation capacity that uses germinated seedlings was developed in support of Sub-objective 1A. The assay is being developed as a tool for breeding annual ryegrass with tolerance to acidic soils. Tests with small populations indicated that results with germinated seeds are predictive of adult plant performance and that the trait is heritable from parents to offspring in annual ryegrass. Selection for high chelation capacity may result in a correlated response for increased seedling vigor. More testing is needed to determine if this is the case. The results so far indicate that this assay is valuable for determining ryegrass root exudate chelating activities and that the trait can be improved through selective breeding. Progress on Sub-objective 1B was made by developing a herbicide susceptibility assay and developing populations of annual ryegrass for selection experiments and breeding. Herbicide susceptible annual ryegrass is needed to make using it as a cover crop easier. In vitro leaf assays using a large range of glyphosate concentrations were unable to discriminate between herbicide susceptible and tolerant selections. 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 assay was used to evaluate and select annual ryegrass plants for tolerance and susceptibility. Each group was subsequently transplanted into isolation blocks for intermating and seed production. Seed from the ‘tolerant’ and ‘susceptible’ selection groups will be used to conduct experiments determining the response from selection for herbicide susceptibility. Continued progress was made towards Sub-objective 2A on the evaluation of grasses for the presence of two different strains of Barley yellow dwarf virus (BYDV MAV and PAV) and Cereal yellow dwarf virus (CYDV), the causal agents of barley yellow dwarf (BYD) disease. Field plots were maintained and the border areas of the plot were planted with Alba, a BYD susceptible barley, to maintain BYD disease pressure. Samples were assayed for CYDV and two different BYDV strains to determine infection types and levels in the different cultivars. 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. Stem rust caused by the fungus Puccinia graminis (pgl) is a destructive disease of perennial ryegrass grown for seed. Field experiments with perennial ryegrass clones and collection of pgl isolates were conducted at three sites in 2019 and two sites in 2020. More than 30 perennial ryegrass clones were transplanted to replicated field experiments and evaluated for the rate of reproductive development and stem rust severity when the disease occurred. Disease data will be used with data collected from previous field experiments to study the stability of resistance across Pacific Northwest production sites. More than 300 unique collections of pgl, referred to as isolates, have been collected from disease resistant plants for use in genetic diversity studies with simple sequence repeat (SSR) molecular markers. Amplification of SSR markers by polymerase chain reaction is needed to test their utility in research. This step has been completed for more than 70 SSR markers and 36 SSR markers have been tested with multiple pgl isolates to begin to identify genetic differences. 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. There are currently no management tools to control the fungus Epichloë typhina that causes choke, a disease which can result in yield losses of up to 30% in orchardgrass seed production fields in the Willamette Valley Oregon, where most orchardgrass seed for the United States is produced. Development of choke resistant orchardgrass germplasm is a long-term approach to disease management and is the focus of Sub-objective 2B. Data on flowering and presence of choke was collected from previously established replicate trials containing progeny from potential choke resistant maternal lines and susceptible and resistant control cultivars. Plants that were preinoculated with Epichloë typhina at the seedling stage were evaluated for the presence of flowers, choked heads, and mixtures of choke/flowers to identify germplasm that may be able to escape expression of choke symptoms when infected with Epichloë typhina. Additionally, three Barlego seedlings inoculated with Epichloë typhina were tiller propagated to produce 90 clones for each seedling, which were used to establish a field plot to facilitate testing for disease management strategies while minimizing the confounding effects of genetic variation and infection rates. Project scientists, as part of the National Turf Grass Sequencing Initiative, continued progress towards developing molecular resources for perennial ryegrass to address Sub-objective 4A. Plants previously selected for the perennial ryegrass transcriptome sequencing project were subjected to drought and samples from control and drought-treated plants were prepared and submitted to Omega Bioservices for RNA-sequencing. In collaboration with scientists in Logan, Utah, the transcripts have been assembled, and the annotation and expression analysis are currently in progress. An individual Lolium perenne cv Manhattan plant, which is true to type phenotypically with strong vernalization and photoperiod requirements, has been selected for whole genome sequencing. Flowering stalks, control and stressed root and leaf tissue has been collected from this plant for RNA-sequencing with the objective of developing a more complete transcriptome for perennial ryegrass. Grasses used as forage or turf are challenged constantly by a variety of stresses, from wounding via cutting or grazing to high temperatures and drought conditions. Plants must be able to respond to these various stresses to survive. A better understanding of how plants respond to these stresses will be useful in breeding grasses for improved performance in different environments. To determine how plants respond to wounding, project scientists, in support of Sub-objective 4B, examined genes present in wounded plants and compared them to the genes present in unwounded plants. They identified 9,413 genes that were induced in response to wounding and 7,704 genes with decreased expression within 24 hours of wounding. Some of these genes encoded for proteins that may enable the plant to sense wounding and respond to it. Some may act as signals to warn the rest of the plant or even other plants nearby of danger, and some probably act to alter growth and mediate other stress responses. These identified genes provide a valuable molecular resource that 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. Another major stress that grasses are exposed to is drought stress, which often occurs concurrently with heat stress. A similar approach was used to identify genes involved in the heat/drought response. Since plants can't escape or change their environment, the analysis of genes differentially expressed between control and drought/heat treated plants should give researchers a better understanding of how grasses respond to survive heat/drought stress. Strategies to increase stress tolerance in crops will become increasingly important for global food security with increasing population and climate variability. In response to stresses, plants utilize regulatory proteins to change the expression of stress responsive genes to mediate a stress response. Identifying these regulatory proteins and understanding their role in stress responses could provide potential avenues for developing stress tolerant crops. As part of Sub-objective 4C, a gene encoding a regulatory protein, which was previously shown to be highly expressed in response to multiple stresses, was overexpressed in a model grass species to determine its potential for improving stress tolerance. Unexpectedly, plants overexpressing this regulatory protein were much smaller than control plants, but this difference in stature was less pronounced when plants were grown under salinity conditions. Plants overexpressing this regulatory gene exhibited gene expression changes that were similar to those observed in model plants exposed to salinity. This suggests that this transcription factor is integral in abiotic stress responses, and that overexpressing this protein is placing the plants in a constant state of stress response, which probably contributes to the plants' reduced stature.

1. Analysis of unintended effects of overexpressing the bZIP26 transcription factor in Brachypodium. Improving stress tolerance in crops will become increasingly important for global food security with increasing population and climate variability. Understanding the strategies plants utilize to respond to stresses and how these responses are regulated is important for developing stress tolerant crops. Researchers in Corvallis, Oregon, overexpressed a gene encoding a regulatory protein that was previously shown to be elevated in response to multiple stresses in order to determine its potential for improving abiotic stress tolerance in grasses. Unexpectedly, plants with high levels of this regulatory protein were reduced in stature and displayed altered gene expression patterns similar to that observed in control plants exposed to salinity stress. These results support the importance of this gene in the stress response, but also reveal the necessity of looking for unintended consequences that can occur when manipulating genes for improved stress tolerance. In the long term, information on the role of regulatory proteins in stress responses will facilitate modification of crops to improve their ability to survive environmental stresses.

Review Publications
Martin, R.C., Kronmiller, B.A., Dombrowski, J.E. 2020. Transcriptome analysis of responses in Brachypodium distachyon overexpressing the BdbZIP26 transcription factor. Biomed Central (BMC) Plant Biology. 20:174.
Dombrowski, J.E., Kronmiller, B.A., Hollenbeck, V.G., Martin, R.C. 2020. Transcriptome analysis of wounding in the model grass Lolium temulentum. Plants. 9(6):780.