Graduate Thesis Or Dissertation
 

The Effects of Ploidy Manipulation on ‘I3’ hemp

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/zg64tv13g

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  • Hemp is a dioecious, annual, flowering plant in the Cannabaceae family. It has been used for thousands of years to produce textiles, essential oils, and medicines. The overall purpose of these experiments was to propagate the cannabinoid-free cultivar, ‘I3’, and follow up by inducing polyploidy in the ‘I3’ hemp via chromosome doubling, and then to determine if/how the chromosome doubling process caused any polymorphisms or somatic mutations within the ‘I3’ hemp genome. To accomplish this, we first performed a hemp propagation study with vegetative stem cuttings to determine if propagation environment, rooting hormone formulation, and rooting hormone concentration influenced survival as well as root and shoot quality. We concluded that the talc powder formulation applied at 3000 ppm, or using no rooting hormone for propagation delivered the best results of survival as well as root and shoot quality. To double the chromosomes of the ‘I3’ hemp genome, we soaked the ‘I3’ meristems in chemical baths containing different mitotic spindle inhibitors (colchicine and oryzalin) at different concentrations, and for different treatment lengths to see if we could find an optimal combination of treatments that would allow us to successfully double the chromosome number. We had success doubling the ‘I3’ genome using both in vivo and in vitro methods. To compare the ‘I3’ diploid genome to the tetraploid genome, we used comparative genomics. We used the hifiasm assembler to assemble our genomes, and then used a variety of bioinformatic tools such as BUSCO, QUAST, minimap2, and others to help us determine if ploidy manipulation provides a “perfect” genome duplication, or if there were any somatic mutations in the tetraploid genome that occurred from the chromosome doubling treatment. We used CBDRx as a reference genome to help align all the ‘I3’ scaffolds to CBDRx chromosomes, and then the diploid and tetraploid genomes were compared. After comparing the ‘I3’ diploid and tetraploid genomes, we were able to conclude that the chromosome doubling process does not result in a “perfect” duplication. We found variants in the form of single nucleotide polymorphisms on all 10 chromosomes, as well as observing large gaps in the tetraploid alignments. These gaps could be some sort of repeat insertion, or a mutation, or they could also simply be the result of fragmentation since we are comparing two different qualities of genome assembly. Further research can be done to figure out the exact cause of these gaps in alignments, but polymorphisms were confirmed on every chromosome.
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  • Oregon CBD
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