Location: Floral and Nursery Plants Research2019 Annual Report
1a. Objectives (from AD-416):
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. 3. Determine the impacts of stress (heat and drought) on Danthonia gene expression and reproductive biology. (NP215 2D)
1b. Approach (from AD-416):
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.
3. Progress Report:
Sequencing of the internal transcribed spacer 1 and 2 regions of the 5.8S rRNA gene and the analysis of ten SSR loci from 27 A. hypoxylon isolates collected at five different locations has been completed. The results indicate that two distinct fungal types exist in natural habitats. Five single nucleotide polymorphisms in the ITS 1 spacer and all ten SSR loci can discriminate the two fungal types. The results show that sexual reproduction is not occurring in the fungus. Furthermore, the random distribution of the two fungal types within locations is similar to the genetic diversity exhibited by the host grass D. spicata.