Location: Forage Seed and Cereal Research Unit
2023 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
In support of Sub-objective 1B, diverse genetic elements underling glyphosate resistance and susceptibility in annual ryegrass were investigated. We have implemented molecular biology assays to identify common genetic sources of glyphosate resistance. These include an assay to differentiate amino acid substitutions in the glyphosate target enzyme EPSPS and an assay to measure an increase in genomic copy number of the gene encoding EPSPS. These assays are being used to distinguish between annual ryegrass populations with common and uncommon sources of glyphosate resistance. Investigating uncommon sources of variation in glyphosate resistance can help us to breed for and maintain annual ryegrass with increased susceptibility and reliable, glyphosate-induced termination.
Selection of orchardgrass plants that are resistant to the disease choke has been advanced in support of Sub-objective 2B. The choke resistant plants have been divided into two groups based on maturity. These plants were then transplanted into two sets to produce seed under both greenhouse and field conditions. The greenhouse set was vernalized in the growth chamber for six weeks at 4°C then moved to greenhouse at 20°C to flower and set seed. The field set was not vernalized and is being maintained outside of the greenhouse until it can be transplanted to the field in October 2023. The plants will then vernalize and flower under ambient conditions. Once adequate quantities of seed are harvested from the greenhouse or field sets, the choke resistant orchardgrass will be released as two improved germplasms, an early maturing group and a late maturing group. Both germplasm releases will maintain pedigree and passport data by the female parent. Additionally, specific plants within each maturity group have been identified and will be polycrossed in the 2023/2024 field season to develop a first generation synthetic to be released as a variety.
We determined the genetic diversity of Puccinia graminis f. sp. lolii (Pgl), the fungus causing stem rust in perennial ryegrass. This work is in support of Sub-objective 2C. Genetically variable plant pathogens may require multi-tactic disease control approaches and little is known about the genetic variation of Pgl. Twenty-one simple sequence repeat 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 21 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.
In support of Objective 4, we have begun testing tissue culture protocols for the model grass Brachypodium distachyon and crop species Lolium multiflorum (annual ryegrass). Plant embryos have been converted successfully to undifferentiated callus and back to differentiated plant tissues for both species. Tissue culture is one of the first necessary steps to perform Agrobacterium-mediated transformation and gene editing. These tools together will be invaluable for understanding gene function and for improving crop traits via molecular methods.
In support of Sub-objectives 4A and 4B, we continued to work towards developing molecular resources for perennial ryegrass as part of the National Turf Grass Sequencing Initiative. In collaboration with scientists in Logan, Utah, the perennial ryegrass cv Manhattan reference genome has been assembled and annotated. 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. Using previously generated perennial ryegrass drought response transcriptomic data, we will be able to assess which LEA genes are differentially expressed in response to drought and how they relate to genetic pathways regulating abiotic stress.
We have also begun to sample annual ryegrass varieties to explore the genomic diversity within this species. Four samples from each of three annual and three biennial varieties have been submitted for Illumina sequencing. Our 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 will be particularly valuable for understanding the variation in flowering time within this species. This work also supports Objective 4 by building resources that establish the inherent molecular variation within this grass species.
To further advance abiotic stress resistance in cool-season grasses, we have initiated the first steps in a genomic selection (GS) program for low temperature and high temperature tolerance, among other traits, in tall fescue and perennial ryegrass. This includes developing multi-stage phenotyping protocols for assessing cold tolerance as it relates to frost, freeze-thaw, and ice-encasement as well as heat resistance traits such as drought, salinity, and dormancy under controlled growth chamber and greenhouse conditions. Additionally, starting germplasm has been collected based on phenotypic variation in key traits for use as parent material in the GS program. Furthermore, this material will serve as the basis of the tall fescue and perennial ryegrass breeding program and therefore specific cross combinations will be made based on the genetic diversity of the parent material. This will be determined by genotyping-by-sequencing (GBS) and the first round of GBS in currently underway.
Accomplishments
