Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-033-08-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2022
1) To cross deacclimation resistant canola to deacclimation sensitive winter lines and elite spring types in order to both confirm the role of previously identified deacclimation loci, and to determine if we can improve survival of susceptible elite spring and winter varieties. 2) Continuation of the previous plan to characterize the function of target genes identified by GWAS.
Perform selected crosses to both confirm the role of previously identified deacclimation loci, and to determine if we can improve survival of susceptible elite spring and winter varieties. We will perform crosses between winter canola lines ARS261 and ARS233, ARS261 and ARS246, and ARS229 and ARS036 as well as between lines ARS229 and two spring lines with low freezing tolerance as demonstrated through the work of Dr. Danielle Fiebelkorn in Dr. Rahman’s collection. A portion of the resulting F1 seeds (minimum of 12 per cross) will be tested for their ability to withstand deacclimation conditions and freezing tolerance using previously defined conditions for these stresses. The remaining seeds (and any surviving F1 plants) will be grown and allowed to self-pollinate to generate F2 seeds. The resulting F2 seeds from each of the 5 crosses will separately be allowed to self to produce segregating F3 families. 12 individuals from each family will be grown and tested for their ability to withstand freezing stress prior to and following deacclimation using established protocols. F2 populations showing high tolerance to freezing and/or resistance to deacclimation will be donated to Drs. Rahman and Stamm as well as anyone else who is interested in them upon request and acceptance of appropriate material transfer agreements. Likewise, families that appear to be strongly segregating for either freezing tolerance and/or deacclimation resistance will be selected for mapping of the traits using GBS markers identified by skim sequencing and comparisons to SNP populations identified in the parents from the original GBS mapping on our diversity panel or the panel from Dr. Rahman from which the parents were selected. Continuation of the previous plan to characterize the function of target genes identified by GWAS. Target genes will be screened using arabidopsis that contain knockout mutations of the target genes and testing their impact on deacclimation. It is possible to simply order lines of arabidopsis from the publicly accessible arabidopsis mutant collections that have mutations in most genes, and we have already developed methods for following and screening lines for differences in deacclimation processes in arabidopsis. Genes that impact the deacclimation process will be subjected to further analysis. To confirm the role of selected target genes in cold deacclimation and associated freezing tolerance of winter canola, we will use gene editing CRISPR technology (Bortesi and Fischer 2015).