Location: Floral and Nursery Plants Research
2023 Annual Report
Objectives
1. Enhance marker resources and diversity assessment in the genus Danthonia. (NP215 2D)
Sub-objective 1.A: Utilize induced mutations to study D.spicata reproductive biology.
Sub-objective 1.B: Evaluate native Danthonia species genetic diversity and ploidy.
2. Characterize the phytobiome of native and newly established Danthonia populations and correlate them with biotic and abiotic stress resistance. (NP215 2D)
Sub-objective 2.A: Evaluate the genetic diversity of Atkinsonella hypoxylon associated with D. spicata.
Sub-objective 2.B: Utilize culture-dependent fungal isolation to identify the endophytic fungal populations of D. spicata from diverse locations.
Subobjective 2.C: Determine the contribution of the Danthonia phytobiome on ecosystem services in the DC metropolitan urban environment.
3. Determine the impacts of stress (heat and drought) on Danthonia gene expression and reproductive biology. (NP215 2D)
4. Characterize the phytobiome of turf species and determine their role in disease resistance in the warm humid climate of the mid-Atlantic U.S. The research will investigate the contribution of the turf phytobiome to ecosystem services and disease resistance.
Approach
The native grass Danthonia spicata has a number of characteristics that make it well suited for use as a low input turf in the U.S. mid-Atlantic region. The primary drawback for utilization is poor seed production potential and very limited genetic diversity. For example, D. spicata seed production suffers from seed shattering, a common characteristic of unimproved grasses, but its lack of genetic diversity combined with its unusual reproductive characteristics do not provide options for improving seed production through conventional breeding approaches.
The unusual reproductive characteristics of D. spicata include anther developmental arrest at a very early stage and precocious seed production without any evidence of fertilization. Progeny plants exhibit extreme uniformity, and SSR markers scored on progeny populations are monomorphic. Although apomixis would be a possible reproductive strategy, apomicts typically exhibit high levels of fixed heterozygosity due to premeiotic embryo formation. Post-meiotic automixis, either through the formation of a restitution nucleus or an endomitotic event, are additional possible mechanisms. To test the various reproductive strategies genetically and to generate variation for plant improvement requires the creation of polymorphisms that can be followed through meiosis; this will be done through mutation breeding.
The genus Danthonia includes a number of species that have been recognized as native grasses of the U.S. The existence of an unusual reproductive biology in the genus and the fact that a number of the proposed species grow in close proximity suggests that some of the species may simply be variants of a single species. One mechanism that would support the current species designations is polyploidy. Currently no data is available on the ploidy levels of the native Danthonia species. A second factor that would support the species designations is large amounts of genetic variation between the described species; this will be tested using Simple Sequence Repeat (SSR) markers.
Atkinsonella hypoxylon (Clavicipitaceae, tribe Balansieae) is an Ascomycete that has been reported to grow epiphytically and endophytically on grasses in the genus Danthonia. Genetic diversity assessments of A. hypoxylon have been completed; however, they were conducted using isozyme technology and no Internal Transcribed Spacer (ITS) data was obtained to confirm isolate taxonomy. The genome sequence of A. hypoxylon has been determined, making Simple Sequence Repeat (SSR) marker development possible; currently our lab has approximately 20 functional SSR primer pairs that have been tested on isolates from 4 locations. Additionally, all isolates we have examined exhibit multiple loci with 2 alleles. This multiallelic state in what is expected to be haploid hyphae has also been reported in Epichloe festucae and could be due to multistrain infections of the plants. ITS cloning will be utilized to determine if Danthonia plants harbor multiple symbiotic fungi.
Progress Report
Danthonia has almost 8-10 times more tolerance to drought stress than other turfgrasses such as bentgrass. We began studies to compare metabolic changes of Danthonia vs. bentgrass in response to drought stress in order to understand the underlying mechanism of drought tolerance in Danthonia.
Previous reports indicate that the enhanced drought tolerance in Danthonia might be related to the plant’s interaction with an epiphyte, Atkinsonella hypoxylon. We began to quantify the overall level of the fungus at different locations in the plant and at different growth stages using dd-PCR to investigate the relationship between drought tolerance and the presence of this epiphytic fungus.
We used molecular markers to identify highly crossable plants of creeping bentgrass and colonial bentgrass and developed an F1 interspecific hybrid bentgrass population using these plants, and, with collaborators in Logan, Utah, generated a genetic map of the population using Genotype by Sequencing (GBS). Previously, interspecific hybrids between creeping and colonial bentgrass showed enhanced disease resistance to dollar spot. This new hybrid population will be utilized to study the genetics of important characteristics such as high yield, enhanced disease resistance, drought tolerance, and different morphological traits, such as stolon vs rhizome production.
To investigate the relationships in bentgrass species between winter dormancy, desiccation, and growth of stolons (creeping bentgrass) vs. rhizomes (colonial bentgrass), we conducted winter drought studies. We found that most rhizomatous colonial-type hybrids were susceptible to drought, which suggests that enhanced drought tolerance of colonial bentgrass is suppressed by weakness in winter dormancy through shorter daylength and low temperature. More investigation is needed; however, we found several hybrids exhibiting both rhizomes and stolons with enhanced drought tolerance. We are currently evaluating stoloniferous types, rhizomatous types, and the combination type to determine how these phenotypes correlate with stress symptoms.
In addition, we developed an artificial intelligence (AI)-based automatic image analysis system through Machine Learning/Deep Learning to facilitate the efficient collection of phenotypic data to accelerate the development of superior turf germplasm. This system can automatically (1) capture images, (2) segment an image containing numerous plants in an image into sub-images with one plant per image, (3) label each image with the corresponding genotype information, (4) quantify stress symptoms, and (5) classify them based on their performance. This platform is being used to map quantitative trait loci, identify candidate genes, and develop new molecular markers in turfgrass.
Accomplishments
1. New bentgrass hybrids created. Creeping bentgrass is widely grown on golf course greens, tees, and fairways as it produces a dense, uniform playing surface with excellent recovery from damage. However, it requires high maintenance such as frequent irrigation, fungicide and fertilizer applications, and core cultivation with topdressing to reduce organic matter buildup. Colonial bentgrass generally has better disease resistance, drought tolerance, wear tolerance, and requires less frequent core cultivation and top dressing because it spreads primarily through underground rhizomes rather than above ground stolons. In an effort to combine the desirable traits of both species, ARS scientists in Beltsville, Maryland, created hybrids between creeping and colonial bentgrass. These hybrid plants have promise as new turf types that will reduce golf course maintenance requirements.
2. Novel approach for analyzing genetic diversity in outcrossing grasses. Interspecific hybridization can result in improved plant performance by masking deleterious genes and combining desirable characteristics from diverse species. Knowledge about the species’ genetic relationships and variability is often used to increase the success of interspecific hybridizations. However, in turfgrass, high levels of outcrossing and polyploidy can make it difficult to assess the level of genetic diversity to plan successful crossing experiments. ARS researchers in Beltsville, Maryland, developed a new approach to estimate the level of diversity in bentgrass species using DNA markers and novel data scoring methods. The data generated from this approach was used to identify individual bentgrass plants that would be most likely to successfully cross to produce interspecific hybrids.
Review Publications
Warnke, S.E., Barnaby, J.Y. 2023. Genetic diversity of Colonial bentgrass Agrostis capillaris based on simple sequence repeat markers and high-resolution melt analysis with haplotype scoring. Crop Science. https://doi.org/10.1002/csc2.20943.
Cortes, A.J., Barnaby, J.Y. 2023. Harnessing Genebanks: High-throughput phenotyping and genotyping of crop wild relatives and landraces. Frontiers in Plant Science. 14:1149469. https://doi.org/10.3389/fpls.2023.1149469.
