Strategies to Modulate Recombination in Large Genome Crop

Background
In barley (and wheat), substantial proportions of the chromosomes are inherited together as a large linkage block, preventing the generation of novel gene combinations and useful variation that could be exploited in breeding programs. In these crops, the distribution of meiotic crossover events is skewed toward the telomere regions meaning that up to half of the genes rarely if ever recombine. This project aims to generate lines with increased recombination in low recombinogenic region, which could be use in breeding program to produce new barley variety, with better agronomic traits, better resilience to stress such as pest and disease.
Aim/Objectives
Meiotic recombination is a highly controlled process to ensure the species’ genome integrity. Over the years, recent findings have described anti-crossover factors, that once downregulated, could increase meiotic recombination in plants. The helicase SRS2 has been found to increase recombination in yeast by modulating strand invasion by the protein RAD51 Similarly in Arabidopsis, SRS2 controls RAD51 intermediates and restricts recombination. We identified one SRS2 orthologue in barley on chromosome 2HS, highly expressed in meiotic tissue. Little is known about UrVD helicase in plants despite their role in chromatin remodeling and regulation of recombination. Therefore, the aim of the project will be to generate barley plants with an altered expression of SRS2 and monitor the effect on meiosis and recombination. In Arabidopsis, two classes of Crossover co-exist. The first class of crossover (85% CO) are subject to ‘interference’ a process that constrains the position of neighbouring recombination events. The second class (15% CO) is supposedly not interfering, and allows a more random distribution of crossovers, in particular in pericentromeric regions. Studies hint that the proportion of class I crossovers is higher in barley, effectively restricting the distribution of the CO near the telomeres . Since, SRS2 has been shown to play a role within the Mus81/mms4 pathway (calls II), the second aim of the study would be to study the class II pathway though comparisons to our already characterized mutants, such as des10 , defective for class I crossovers.
Methods/Approach
The project will start by generating/screening for plants mutated for SRS2 that will be used for this study. This will constitute the priority for the first year of the PhD and this will involve a number of specific skills such as:
A- Cloning and transformation: Cloning SRS2 genes in barley and generate transgenic plants with our in house CRISPR/cas9 vector for barley (Dr A. Barakate and FUNGEN).
B- Antibody production: Protein expression by using the SRS2 previously cloned, and send to immunization (eg: Dundee Cell Product or more competitive supplier)
C- Tilling Screen: PCR or exome capture screen of Golden Promise and Optic EMS population to generate a series of alleles for SRS2. Different alleles could lead to different genotypes.
D- Cytology: The work will involve DNA in situ hybridization, immunochemistry and BrdU/EDU time course. The first year will be on wild type Golden Promise (and Optic)y, in order to build up experience and establish a reference for the mutant analysis. From year 2, mutants and wild type will be characterized in parallel.
E- Genetic Segregation: From year 2, the student will make crosses between the mutants line in different backgrounds if possible to create a segregating F2 population. The student will identify polymorphisms between the parental line in order to design a genetic marker assay and be able to assess recombination genetically under the influence of SRS2 or not. An alternative will be to cross a mutant line to a standard cultivar and study the recombination patterns in the F3.
F- Gene network and validation: First, the student will complement both Arabidopsis (JHI) and yeast (Sheffield) with the barley orthologue for gene validation. Moreover, we propose to do a screen to rescue the synthetic lethality in S. cervisiae sgs1srs2 double mutants by expressing plant cDNA library. This has the potential to identify plant genes that can functionally substitute to either srs2 or sgs1 bit are difficult to determine from bioinformatic analysis alone. Depending on the success of the interspecific screen we would also consider a yeast-two hybrid screen using HvSRS2 to identify novel interactors. Data can be compared to the meiotic RNA-seq database currently being developed in our lab and against an srs2 RNA-seq reference dataset developed in this project.