STRESS RESPONSES OF THE FLAX GENOME: ACTIVATION OF TRANSPOSABLE ELEMENTS, DEFENSE GENES AND GENOME RESTRUCTURING AND DIVERSIFICATION.

• Author / Creator

  Galindo González, Leonardo M

• Transposable elements (TEs) are DNA sequences that can move in the genome (transpose) via a DNA or a RNA intermediate. TEs are abundant in plant genomes and can be activated by stress. Their activity can result in structural and gene expression alterations that increase diversity within and among species. Canada is a major exporter of flax, which is a source of valuable seed and stem fiber-derived bioproducts. To understand and improve characteristics of seeds and fibers, genomic approaches are now being applied. The sequencing of the flax genome, led by our lab group, showed a landscape with over 43,000 protein coding genes and more than 23% of TE genome coverage. The largest group of TEs were the Ty1-copia elements, and bioinformatic analysis indicated that many of them could still remain active. While these mobile elements are widespread in the flax genome, their influence in diversification, gene expression and genome restructuring is yet to be assessed. In the current study we anayzed members of the Ty1-copia superfamily in flax cultivars, and potential elicitors of TE activation. One of these elicitors (fungal inoculation with Fusarium oxysporum) allowed us to analyze the general defense response of flax to this pathogen which constitutes a threat to flax cultivation. Finally, we designed a reverse genetics methodology to find mutations in genes of interest that could be related to phenotypic changes and used in the future to dissect TE-controlling mechanisms used by the host genome. We first compared flax cultivars using TE-derived molecular markers, and found that retrotransposition events have occurred since breeding began and that TE polymorphisms allowed us to separate flax types. Most TE insertions derived from these polymorphisms fell in close proximity or inside genes, and can potentially alter gene expression. We then tested potential modulators of TE transcription including wounding, fungal extracts, fungal infection, and different plant tissues. The analyses with end-point PCR, quantitative reverse transcriptase PCR, and RNA-seq, showed little evidence that most treatments affected TE activation, but many TE families had high constitutive expression. TE expression across plant tissues resulted in differences that indicate that a better resolution on TE expression modulation can be found when studying meristems and reproductive tissues. While inoculation with F. oxysporum did not alter TE expression, the RNA-seq used to survey TE changes gave additional information on gene regulation upon fungal infection. Most expected defense mechanisms were activated when flax was challenged with F. oxysporum: detection of fungal elicitors, signal transduction cascades, transcriptional reprogramming, activation of defense genes, hormonal signalling, and secondary metabolism modulation. However, the activation of certain genes involved in auxin regulation, cell growth, cell wall expansion, water and nutrient mobilization, plus the repression of major latex proteins, indicated possible manipulation of the host by the pathogen to facilitate infection. Many of the genes found related to plant defense constitute good candidates to analyze relationships between gene expression and disease resistance across flax cultivars. In the meantime, the modulation of unexpected genes opens a door to study cross-kingdom epigenetic manipulation mechanisms (e.g. small RNAs). Finally, we designed a reverse genetics methodology to simultaneously test hundreds of flax mutagenized lines, to discover mutations in genes of interest, using next generation sequencing Ion Torrent technology. Several mutations were found in cell wall and metabolism genes, but with no phenotypic effects. However, this methodology can be applied in the future to detect mutated genes involved in the process of epigenetic modification (e.g. methylation) that results in TE silencing, to test the effects on TE activation.

• Subjects / Keywords

  • Fusarium oxysporum
  • Next-generation sequencing
  • Flax
  • Defense genes
  • Retrotransposons
  • Ion Torrent
  • Transposable elements
  • Linum usitatissimum

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3QV3CG4W

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.