Craig E. Coleman, Department of Plant and Wildlife Sciences
The objectives of this proposal were as follows:
1. Develop single nucleotide polymorphism (SNP) markers for Bromus tectorum (cheatgrass) for use in determining population structure and mapping adaptive traits.
2. Complete a full genome sequence of the Pyrenophora semeniperda fungal pathogen to identify genes encoding important traits related to its pathogenicity on cheatgrass seeds.
3. Complete a full genome sequence of the Ustilago bullata head smut to facilitate identification of virulence genes associated with its pathogenicity on cheatgrass.
Beginning in 2000 students working in my lab developed DNA markers to assess variation in cheatgrass populations as part of a collaborative project with the USDA Forest Service Shrub Lab located in Provo, UT. We identified six simple sequence repeat (SSR) markers that were subsequently used in several important studies describing the structure of populations in the Intermountain Western United States. For the most part, we only used four of the six markers because the remaining two markers provided little additional information about population structure. Because of concerns about the effectiveness of a four-marker system and the robustness of SSR markers, we decided to develop a set of SNP markers that would facilitate a more complete analysis of cheatgrass population genetics. Funds from this MEG were used to develop that marker system. We sequenced cDNA libraries from two different tissues (floral and seedling) of three different cheatgrass accessions using the 454 FLX sequencing platform available in the BYU sequencing center. From the sequence information we extracted thousands of putative SNPs for analysis. We screened over 200 SNPs to identify highly polymorphic loci that could be used in the population analyses. Ultimately, we selected 96 SNP markers that we have used to assess cheatgrass population structure.
We first used the SNP markers to assess outcrossing in cheatgrass populations in the wild and in a common garden setting at the BYU Spanish Fork farm. This study was completed by two undergraduate students, Sudeep Ghimire and Samuel Decker and the work was published in the Journal of Heredity (Meyer SE, Ghimire S, Decker S, Merrill KR, Coleman CE (2012) The ghost of outcrossing past in downy brome, an inbreeding annual grass. J Hered 104:476-490). They determined that outcrossing rarely occurs and that there are certain genotypes and environments more conducive to outcrossing. Later, undergraduate student Desiree Lara initiated a study using our SNP markers to assess the genetic structure of cheatgrass populations in warm desert regions of the Sonoran and Chihuahuan deserts and compare them to populations from surrounding regions. She continued that research as a Masters level graduate student and successfully defended her thesis in December, 2013. Her research will be submitted as a manuscript for publication in the coming weeks. We are holding off publication until we complete another
manuscript describing a comparative analysis of cheatgrass populations from the invaded range of the Intermountain Western United States and the native range of Eurasia. This study also employed our SNP markers and it shows that certain genotypes which have successfully invaded extreme environments of the North American west are found in similar environments of the native range. This work was begun by an undergraduate student, Keith Merrill, who eventually moved into a graduate program. He was assisted by another undergraduate student, Sudeep Ghimire, who is currently in the middle of his Master’s degree program. Another project in which we have used our SNP markers is an analysis of cheatgrass genetics from herbarium samples. BYU Master’s student Desiree Lara obtained herbarium samples from collections throughout the western United States and determined the genotype of those samples using the SNP markers. A manuscript describing her results is currently in preparation.
A full genome sequence for Pyrenophora semeniperda has been assembled and annotated. The work was completed under the direction of M.S. student Marcus Soliai with assistance from several undergraduate students including, Paul Bodily, Russ Hermansen, David Elzinga and Aaron Hart. A report of this work was recently published (M.M. Soliai et al. 2014. De novo genome assembly of the fungal plant pathogen Pyrenophora semeniperda. PLOS ONE 9:e87045.) Since the completion of the genome sequence of a single isolate of the fungus we have subsequently resequenced the genome from 12 additional isolates using funding from the Utah Department of Agriculture and Food. This work was done by undergraduate student Julie Henry.
The work on Ustilago bullata reached a dead-end because of problems with matching fungal isolates with host ecotypes. Although we have genome DNA sequence data for this fungus, we have not completed a full assembly or annotation on that data. Resources dedicated to U. bullata research were diverted to P. semeniperda because of the greater promise that it shows as a potential bio-weapon against the spread of cheatgrass across its range in western North America.
In summary, the funds provided under this MEG resulted in the publication of one manuscript related to the development of SNPs in cheatgrass with two more publications in preparation. A second manuscript has been published reporting the details of the assembly and annotation of the P. semeniperda fungal genome. These funds supported mentored research for ten undergraduate students who are listed below. All of these students were vital contributors to the research which all took place in the Genetics Great Lab at Brigham Young University.
The budget of $20,000 was spent on student salaries ($8,000), supplies including reagents for DNA extraction and sequencing ($10,000) and travel to scientific meetings ($2,000) to present the results of our research.