Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-033-015-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 28, 2021
End Date: Oct 31, 2023
Objective:
1. Genotype Introgression Lines (ILs) of canola to confirm the mapped 26-Kb heterotic region on chromosome A10 involved in increased grain yield;
2. Sequence the 26-Kb region on chromosome A10 using a primer walking method;
3. Functionally evaluate the roles of the candidate genes identified within the 26-Kb heterotic region.
Approach:
1. Several ILs of canola will be genotyped to confirm the mapped 26-Kb heterotic region on chromosome A10, based on the analysis of genotyping and current phenotyping (grain yield) data.
2. The 26-Kb mapped heterotic region will be sequenced using primer-based sequence walking until enough DNA information is obtained to identify candidate heterotic genes.
3. CRISPR-Cas9 technology will be applied to target the unique sequences present in candidate genes for the generation of canola knockout mutants.
4. For CRISPR-Cas9 work, paired guide RNAs will be designed based on the sequence of candidate genes, cloned into the intermediate vector pCBC, and further cloned into the pCAMBIA-derived binary vectors with a hygromycin-resistance gene as a selectable marker using the protocol for dicot plants.
5. Recombinant plasmids confirmed by sequencing will be used to transform a Chromosome Segment Substitution Line (CSSL) for functional characterization of candidate heterotic genes.
6. CRISPR-Cas9-mediated genome editing will be confirmed by PCR and subsequent sequencing of the PCR products from T1 lines.
7. Transgenic lines, with confirmed mutations, will be used to test for heterotic effect based on phenotypes of offspring resulting from crossing genetically modified CSSL and Westar.
