Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-031-09-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2015
End Date: Dec 31, 2019
The general objectives of this project are: 1. Functional characterization of potential candidate resistance genes to SSR in B. napus using CRISPR/Cas9 system. 2. Development and validation of molecular markers linked to S. sclerotiorum resistance in canola. 3. Functional characterization of S. sclerotiorum genes associated with pathogenicity on canola.
To achieve the first objective of this proposal, we intend to conduct targeted gene mutagenesis using the CRISPR/Cas9 system, which is an established protocol to generate mutations in target genes. Mutations will be confirmed using standard protocols that combine restriction enzyme site loss assays and cloning and sequencing of mutants. Gene role during infection will be determined using standard inoculation techniques and comparing results to that of wild type. To achieve the second objective, we propose to develop cleave amplified polymorphic sequence (CAPS) markers. These markers are easier to detect using inexpensive equipment. Once markers are developed, we will validate them on canola breeding populations derived from NEP63 and others. To achieve the third objective, we will use an established protocol to knockout target genes identified in a previous project funded by the Initiative. This protocol involves transformation of fungal protoplasts using constructs containing the hph gene as selectable marker. PCR and southern hybridizations will be conducted to verify proper gene replacement has occurred. Mutants will be inoculated on canola plants to determine the effect of gene disruption. The research proposed here contributes to fulfill objectives identified in the Strategic plan of the Sclerotinia Initiative.