EVALUATION AND DEVELOPMENT OF RESOURCES FOR GENOME EDITING AND GENETIC ENGINEERING IN SOLANUM TUBEROSUM

Gene editing has emerged as the most precise method used today for validating gene function and introducing traits to improve crop quality. While a powerful tool when available, its broad applicability in plants is limited by highly genotype dependent methods and inefficient editing systems. The standard method is contingent on the success of several independent steps including identifying target sequences, introducing reagents into plant cells, efficient on target editing activity, recovery of edited cells as whole plants, and the accurate evaluation of target outcomes. If at all attainable, this process is still cumbersome and can span from several months to even years to complete. Developing efficient pipelines for gene editing is an asset for studying or improving any plant species. This dissertation aims to address limitations to gene editing in potato (Solanum tuberosum) through evaluation and optimization of germplasm amenability, development of genomic resources, and a comprehensive analysis of on and off target editing outcomes.First, potato germplasm was evaluated for attributes useful for gene editing including transformation capability, regeneration efficiency, tuber quality, and fertility. This study identified three diploid clones that fulfilled these requirements, 1S1, MSEE737-05 and UW-W4. Second, a genomic resource was developed for the highest achieving clone from the prior study, 1S1. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly containing 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. The alternate haplotype of 1S1 was then deduced through variant analysis using Illumina reads. This study also improved 1S1’s self-fertility response through bud pollinations and developed genetic resources for viral vector gene editing. Third, a whole genome evaluation of genome editing outcomes using CRISPR/Cas9 was conducted for the clonally propagated diploid potato (Solanum tuberosum Grp Phureja, 2n=2x=24) and the seed propagated diploid with three similar subgenomes, camelina (Camelina sativa, 2n=40). In potato, members of the polyphenol oxidase (PPO) gene family were targeted in the clone DRH195, resulting in seven stable transformants with targeted edits in at least one allele of PPO1, PPO2 or PPO3. In camelina, nine transformants targeting fatty acid desaturase 2 (FAD2) were obtained from a previous study (Jiang et al. 2017). Reference based variant analysis using ~55-60X coverage whole genome sequencing reads for each event was used in the evaluation of on and off target editing outcomes. The results of this study indicated negligible off-target editing compared to spontaneous or tissue culture induced variation. Together, this research provided solutions to several of the main challenges faced in gene editing of plants by identifying germplasm amenable to these techniques, optimizing transformation conditions, and complimenting them with genomic resources to facilitate identifying targets and evaluating specificity.