Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-033-004-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Apr 2, 2019
End Date: Mar 31, 2022
Objective:
The overall goal of this project is to identify genes controlling freezing tolerance in camelina that can be used to improve this important agronomic trait in winter and/or spring camelina and other cruciferous cover crops and thus improve the weed control capacity of these crops. To do this, we will: 1) Utilize recombinant inbred lines (RIL) derived from crosses between freezing sensitive cultivars CO46 and the freezing tolerant cultivar Joelle, as well as near isogenic lines (NIL) developed from back cross lines of the freezing tolerant F2 offspring to the freezing sensitive CO46 parent. 2) We will genotype the resulting RILs and NIL using GBS or related technologies. 3) We will phenotype the resulting RILs in regards to freezing tolerance and map the loci controlling these traits. 4) We will use a bulk segregant whole genome next generation sequencing approach on the RIL and NIL populations to identify variants and genomic regions associated with freezing tolerance. 5) We will identify putative genetic variants in genes underlying these differences. 6) We will develop testable hypotheses for future functional genomic analyses.
Approach:
We have developed a recombinant inbred lines (RIL) population of nearly 250 individuals that are segregating for freezing tolerance and that have been confirmed by molecular markers as a result from reciprocal crosses between CO46 (spring type) and Joelle (winter type). These are currently undergoing or have undergone selfing by single seed descent for 5 generations using both field and greenhouse areas to grow and produce seeds. We have access to 3 large walk-in growth chambers capable of vernalizing up to several hundred plants each and three greenhouse wings to assist in advancing lines requiring vernalization, and we expect to be able to grow 3-4 generations per year. These will be advanced for at least 7 generations prior to expected funding. Likewise, we expect to have a NIL population back crossed at least 4 or 5 generations (currently at BC3).
We will genotype these advanced Recombinant Inbreed Line (RIL) populations using GBS or related technologies using contracted or in-house resources to identify 10K-30K SNP and short indel markers. Because the freezing tolerance trait is both simple (one or 2 major genes) and because the trait is dominant, we will also perform whole genome sequencing on bulked segregating pools from these populations to identify specific markers and genomic regions associated with the trait.
We have previously established protocols that can easily distinguish the differences in freezing tolerance between the parental lines. We will use these protocols to phenotype each RIL and NIL families with a minimum of three replications to ensure consistency and accuracy of the results.
Standard QTL analysis techniques (including iterative false discovery statistics) will be used identify genetic regions associated with any of the measured phenotypes that segregate with high quality SNP markers. We will utilize several open source or proprietary mapping programs to identify loci.
Draft sequences of the camelina genome, and subsequent guided assemblies from the two parent genomes (in hand) will be used to identify genes and sequence differences within the QTL intervals. This information will be used to predict the genetic variants underlying each QTL.
