Location: Weed and Insect Biology Research
Project Number: 3060-21220-033-025-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: May 1, 2023
End Date: Apr 30, 2027
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 numerous agronomic traits such as seed size, flowering time, days to maturity, freezing tolerance, growth in the cold, root architecture, and weed suppression - among others. Several genetic loci associated with these traits have already been mapped, and target genes have been identified. This project aims to functionally test target genes mapped to these traits and further characterize the genetics associated with them. The first objective of this work will be to use CRISPR technology to knock out target genes in the winter and spring biotypes to functionally test the role of those genes in the traits they are associated with. The second objective will be to further characterize differences in weed suppressing capabilities of winter and spring camelina and to genetically map traits impacting weed suppression capabilities in 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 four different target 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. Likewise, similar CRISPR knockouts of target genes associated with any of the other traits will be identified. 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 two 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.
