Exploring Arabidopsis Molecular Insights, Brassica Napus AHL Genome-wide Analysis, and Genotype Stability Assessment: A Multi-Pronged Approach to Enhance Canola Traits

Shahbaz N Ahmed

doi:10.7273/000006320

This dissertation presents a multi-faceted investigation approach to shed light on the AHL26 (AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED #26) role in Arabidopsis growth and development. To expand its scope, the genome-wide analysis of the AHL gene family was conducted in Brassica napus to provide insights into their conservation and diversity within this economically important specie. Furthermore, winter-type Brassica napus genotypic stability assessment was studied to facilitate the identification of winter tolerance lines, which is crucial for enhancing crop resilience. Collectively, the core objective of this multi-pronged approach was to provide answers to the overarching question of improving plant growth habits at various levels. The first research chapter of this dissertation talks about understanding the AHL26 gene function in Arabidopsis involving a versatile molecular biology toolkit. Leveraging the CRISPR-Cas9 technology, gene overexpression strategies, gene dominant-negative analysis, gene expression, and transcriptome profiling, we were able to unravel the mechanism governing the AHL26 gene role in controlling seedling growth and flowering initiation. Building on the AHL26 gene exploration work using CRISPR-Cas9 technique, my research journey took an exciting turn when we noticed the ability of Arabidopsis seedlings to recover after growing on Basta selection media. So with the AHL26 gene function study, I not only unraveled the role of this gene but also developed a novel technique to select Cas9-free mutants. This innovative technique which emerged from our AHL26 gene function study became the basis of my second research chapter. In this chapter, I not only describe the ability of Arabidopsis seedlings to recover from initial Basta shock, but I also demonstrate how this technique can also be applied in other plants, such as canola and wheat. One of the core objectives of my Ph.D. was to bridge the insights gained from fundamental molecular studies in Arabidopsis to economically important field crops such as canola. As Neff lab is transitioning knowledge gained on the AHL gene family from the model plant to canola, my third research chapter talks about the attributes of this gene family in Brassica napus through a comprehensive genome-wide analysis. The information gained from this research study will serve as a preliminary step by allowing the scientists to identify a potential candidate gene with a targeted role. While molecular research brings important insights into the gene and genetic pathways, projects involving this approach span a considerable amount of time. Being a student in the field of crop science, my ultimate goal was to generate information that could benefit farmers in a short period of time. Finally, the fourth research chapter carries out the multi-location field screening study to identify suitable but stable winter-type canola genotypes for the inland Pacific Northwest region.

Exploring Arabidopsis Molecular Insights, Brassica Napus AHL Genome-wide Analysis, and Genotype Stability Assessment: A Multi-Pronged Approach to Enhance Canola Traits

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