Graduate Thesis Or Dissertation

 

Modern Wheat Breeding: Next Generation Sequencing and CRISPR Transformation Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/gb19fc400

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  • Wheat (Tritium aestivum) is an extremely important crop worldwide. It accounts for almost one quarter of the calories consumed each day by more than one third of the world’s population, and is grown over more land area than any other crop. Wheat breeding programs constantly strive to increase or maintain yields while simultaneously improving pest resistance, quality traits, abiotic stress tolerance, nitrogen use efficiency, and more. This must be done while also attempting to address changing environmental factors such as increasing and unpredictable temperatures due to climate change which can affect winter wheat’s vernalization requirement and shift severity and duration of disease pressures. While wheat is a highly diverse crop owing to the many environments in which it is planted, within each growing region it is relatively monomorphic and the introgression of new traits is often difficult. In order to keep up with demands for performance and to adapt the crop to changing environmental factors, modern wheat breeders are turning to new and emerging technologies. The advent of reduced representation sequencing and modern computational capabilities has made marker-trait discovery relatively easy and affordable for most wheat breeding programs. These advances make it possible for breeders and researchers to develop markers relevant to regionally-adapted germplasm which can significantly decrease the time it takes to develop a new cultivar via the use of Marker Assisted Selection (MAS). The emerging field of targeted genetic editing using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is also being explored for its potential role in wheat breeding through the introduction of traits that cannot not be incorporated using traditional methods. The work for this dissertation addressed the need for marker-trait identification by utilizing a population of 196 recombinant inbred lines (RILs) derived from a cross between ‘Skiles’ and ‘Goetze,’ two cultivars with diverse phenotypes. The RILs were genotyped using Genotyping by Sequencing (GBS), and Quantitative Trait Loci (QTL) were identified after phenotyping for the desired traits. After field testing in three locations over two years, two QTL for stripe rust (Puccinia stiiformis) resistance were identified on chromosomes 3B and 3D. A combination of all resistance alleles together outperformed the resistant parent in every treatment. All identified markers are novel and are immediately useful for MAS to integrate extremely high levels of stripe rust resistance into new varieties. The same RILs were also phenotyped for days to heading after varying vernalization treatments. The lines were subjected to either zero, two, four, or six weeks of vernalization at 6°C, then grown in the greenhouse under uniform conditions, and days to heading were recorded. Four QTL on four chromosomes were identified. The QTL on chromosome 5B is likely a facultative allele known to be in Goetze, but not yet characterized in local germplasm. The QTL on chromosome 5D is also likely a facultative allele that was previously unknown to be in this germplasm. Markers from both QTL can be used for MAS to incorporate facultative growth habits into new varieties. The QTL on chromosomes 1D and 3B are potentially associated with poorly-characterized photoperiod genes, and point to the importance of “minor” genes in flowering time that should be tested further. The work for this dissertation also explored the feasibility of incorporating non-transgenic CRISPR transformation into a public breeding program. The use of CRISPR first requires the in vivo testing of desired gene targets to ensure the chromatin state is not inaccessible at the target location. Therefore, a protocol for protoplast isolation and PEG-mediated transformation using CRISPR Ribonucleoproteins (RNPs) was developed to streamline the process and make implementation in any lab possible. This protocol consistently yields high numbers of protoplasts, does not require specialized equipment, and returns clear positive and negative transformation results for sgRNA targets that are inaccessible in vivo. After testing the gene target functionality in vivo, immature wheat embryos were biolistically bombarded with gold particles coated in CRISPR RNPs, plantlets were regenerated via tissue culture, and transformed individuals were identified with Sanger sequencing, following published protocols. A transformation efficiency of 6.7 percent was achieved at the T0 stage, but subsequent sampling of individual tillers from the fully-grown plants showed the presence of chimeric tissue. This reduced the potential transformation efficiency to below one percent and led to the conclusion that this technique requires considerable time and monetary resources that make it infeasible for use in a public breeding program. It is speculated that successful modern wheat breeding will include a synthesis of all available techniques and technologies for trait improvement such as increased disease resistance, adaptability to unpredictable winter temperatures, and the integration of new traits with genetic editing. This study found novel, useful markers for stripe rust resistance and facultative growth habits in PNW-adapted germplasm that can be used immediately for rapid variety development. While testing CRISPR gene targets in vivo using protoplasts is possible with the developed protocol, the non-transgenic transformation of wheat with CRISPR using currently-published protocols is not feasible. Therefore, while it is clear that marker-trait discovery and MAS are useful and important contributors to modern wheat breeding, targeted gene editing with CRISPR technology requires further study and validation before it can be integrated into breeding programs.
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  • Oregon Wheat Commission
  • ARCS Oregon Chapter Scholar Award
  • Warren E. Kronstad Wheat Research Endowment
  • Oregon Agricultural Experiment Station
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