Location: Weed and Insect Biology Research
Project Number: 3060-21220-033-027-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jan 1, 2024
End Date: Dec 30, 2026
Objective:
Previous studies have identified numerous phenotypic differences between the parents used to develop a 255 Recombinant Inbred Lines (RILs) that are segregating among the RIL populations. These include differences in the flowering-time genes FLOWERING LOCUS C (FLC) that are surprisingly tightly associated with freezing tolerance. This project aims to functionally test the role of FLC genes in freezing tolerance. The first objective of this work will be to use CRISPR technology to knock out each of the three FLC genes in the winter biotypes to functionally test the role of those genes in freezing tolerance. The second objective will be to further characterize differences in weed suppressing capabilities of these mutated camelina.
Approach:
For Objective one, we have obtained a binary vector proven to contain a screenable marker (RED3) that is highly expressed in camelina. This vector will serve as a base for developing an expression vector for the Cas9-enzyme and guide RNAs suitable for knocking out any gene of interest. Once developed, this construct will be modified to express guide RNAs specific for each of the three FLC genes implicated in freezing tolerance differences between the winter and spring camelina biotypes. The sequence of the genes from both parents will be mined from the reference genomes of the parents (which is already available) and guide RNAs will be designed, produced, and ligated into the guide-RNA-expression module of the RED3 CRISPR binary vector. This vector will then be used to transform the two parental lines and full genome sequencing of the progeny (30X coverage for 10 independent lines) will be used characterize the resulting mutations. Mutant lines will be selected based on the number of and severity of the deletions in the target genes, any homogeneous gene copies, and other unanticipated targets of the guide RNAs. Selected lines will then be tested for their freezing tolerance according to or established procedures. For Objective two, we will further quantify differences between the winter and spring biotypes in their weed suppressing and crop-interference capabilities by quantifying the ability of the mutant biotypes to suppress kochia and a commercial sunflower variety. Specifically, 6 pots each containing 4 plants or either spring and winter biotypes will be grown to the 4-leaf stage and a single kochia or sunflower plant at the 4-leaf stage will be planted in the center of each pot. The experiment will be repeated twice each with vernalized and unvernalized camelina biotypes. Above and below-ground biomass of the camelina, kochia, and sunflower plants will be measured after 8 weeks. Root and leaf material from the camelina and the competitor will be collected for subsequent RNAseq analysis.
