Tailoring Camelina Seed Fatty Acid Composition via Fast Neutron Mutagenesis
Date
2022-12-21
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0003-1344-6135
Type
Thesis
Degree Level
Masters
Abstract
Despite its minor status as an oilseed, Camelina sativa is a crop with the potential to reach greater levels of production in Canada due to its unique seed oil composition, relatively low input requirements, and availability of both spring and winter varieties. The limited genetic diversity of the species presents a challenge for camelina improvement through breeding. The aim of this study was to induce genetic variations in camelina’s genome through Fast Neutron (FN) irradiation applied to seeds in a dose gradient ranging from 7.1 to 49.5 Gy. A range of mutations should be caused following FN irradiation including deletions, point mutations, and rearrangements due to DNA damage and subsequent repair. In germination assays of FN-irradiated seeds, FN irradiation had no impact on germination, but higher doses of irradiation resulted in plants with a slower maturity. A FN mutagenesis population was established from irradiated camelina seeds for phenotypic and genotypic characterization. Male sterility and toothed leaf margin of irradiated camelina were observed based on visual observations. Seed fatty acids were analyzed and lineages with altered fatty acid composition, such as high C18:3 and low C20:1, were identified. To study novel genotypes, genotyping by sequencing was applied. An approach was designed and implemented that utilized Illumina sequence technology for generating large amounts of genome sequence data, in conjunction with Oxford Nanopore Technologies (ONT) sequencing for validation. One mutant lineage FN49-38 was characterized whose total very long chain fatty acid composition was 19.6%, a relatively lower percentage than of wild type (23.9%). This lineage carried a genomic DNA deletion of approximately 1.2kb spanning the 5' UTR (untranslated region) and the front half of the FAE1B-ORF (open reading frame) detected through Illumina sequencing, which was validated by ONT sequencing. Furthermore, the accuracy and authenticity of the deletion were confirmed by Sanger sequencing of the cloned mutant gene amplified by PCR (polymerase chain reaction) using specific primers flanking the mutated region. The ONT data was used to review and correct the current annotation of the FAE1 gene on chromosomes 11 and 12. A backcrossing experiment was conducted and confirmed that the deletion was linked to the reduced very long chain fatty acid composition phenotype.
Description
Keywords
fast neutron mutagenesis, FAE1B deletion, fatty acid composition, backcrossing, bioinformatics
Citation
Degree
Master of Science (M.Sc.)
Department
Food and Bioproduct Sciences
Program
Food Science