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The Role of E3 Ligases on the Modulation of Recombination in Cereals


Project Description

Summary
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.

Meiotic recombination is highly controlled to ensure the species genome integrity. Over the years, recent findings have described anti-crossover factors such as FANCM, RECQ4 or FIGL1, that once downregulated, increase meiotic recombination in Arabidopsis. Utilizing the barley mutant collection held at the James Hutton, we mapped a spontaneous mutation, des4 and identified a novel causal gene. Despite semi-fertility, abnormal synapsis caused by unresolved interlocks and sticky metaphases, barley seeds are still produced. Moreover, F3 genotyping analysis revealed that recombination is increased in the distal regions suggesting that this gene is a novel anti-crossover factor that has not yet been described. It appears to be a grass specific gene that we called HvST1 and likely functions as an E3 Ubiquitin Ligase. The causal mutation is due to slippage in a small mononucleotide microsatellite motif before the RING domain that knocks the coding sequence out of frame. In this project we aim to investigate the mode of action of HvST1 in controlling crossovers in barley and wheat.

Proposed approaches for 0-18 months
• Cytological analysis including a time course with EDU and immuno-cytology with antibodies against a range of meiotic proteins will deepen our understanding of the mode of action of HvST1, in particular around the telomeric regions.
• There is some evidence in the literature for interactions between des4 (Hvst1) and des11 mutants. Available Large F2 mapping populations will be used to attempt to clone des11 and we will make double mutants to explore the interaction between des11 and Hvst1
• Only a single mutant allele of HvST1 currently exists. We want to explore whether an allelic series of potentially less deleterious alleles have a sterility and meiotic phenotype. We will use a combination of TILLING and CRISPR-Cas to identify new mutations and characterize their meiotic phenotype.
• Promising mutants will be used to make segregating populations to assess their effect on recombination.
• We have identified mutations in HvST1 in wheat using the tilling populations held at the John Innes Centre and will test for semi-fertility. We have also ordered wheat CRIPSR lines for this gene through the JIC. The student will initiate cytological analyses for the presence of interlocks, telomere fusion and abnormal synapsis as a screening method.
• We will make wheat crosses (including to commercial lines with the 1R/1B translocation) to explore the impact of Hvst1 on genetic segregation and the potential for increasing recombination between homologous and non-homologous chromosomes. This is going to be a collaboration with P. Sourdille group at INRA (Clermont-Ferrand) who have extensive experience in wheat crossing

Possible Approaches for 18-42 months (+6 months for writing)
• Barley: Since this E3 Ubiquitin ligase is a previously uncharacterized gene, the priority will be to define its protein interaction network by using methods such as Y2H, Co-IP and DiGly (ubiquinone) coupled with mass spectrometry. Other assays such as Ubiquitin 48 or 63 could also be established. In addition, as this new gene potentially affects chromatin remodeling, we could envisage establishing a chip-seq method for histone modification. Neither of these methods are fully established in plant meiosis research and will involve considerable development, including taking advice from group leaders at the phosphoprotein facility at the university of Dundee.
• Wheat: The student will continue the characterization of the promising wheat mutant lines, including genetic recombination in collaboration with INRA. All of what is developed in barley can be applied in wheat with the complication of a much-complicated genetic background.

Funding Notes

The studentship is funded under the James Hutton Institute/University Joint PhD programme for a 4 year study period., in this case with the University of Dundee. Applicants should have a first-class honours degree in a relevant subject or a 2.1 honours degree plus Masters (or equivalent).Shortlisted candidates will be interviewed in Jan/Feb 2019. A more detailed plan of the studentship is available to candidates upon application. Funding is available for European applications, but Worldwide applicants who possess suitable self-funding are also invited to apply.

References

1. Isabelle Colas*, Benoit Darrier*, Mikel Arrieta, Sybille Mittmann, Luke Ramsay, Pierre Sourdille and Robbie Waugh. Observation of extensive chromosome axis remodelling during the ‘diffuse-phase’ of meiosis in large genome cereals. (2017) Front Plant Sci. 13;8:1235.
2. Isabelle Colas, Malcolm Macaulay, James D. Higgins, Dylan Phillips, Abdellah Barakate, Markus Posch, Sue J. Armstrong, F. Chris H. Franklin, Claire Halpin, Robbie Waugh, and Luke Ramsay. A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis progression and crossover resolution in the barley desynaptic mutant des10. (2016) New Phytol. 212(3):693-707.
3. Katie Baker, Taniya Dhillon, Isabelle Colas, Nicola Cook, Iain Milne, Linda Cardle, Micha Bayer, and Andrew J. Flavell. Chromatin state analysis of the barley epigenome reveals an ultrastructure defined by H3K27me1 and H3K27me3 abundance. (2015) The Plant Journal 84:111-124.
4. Emma Greer, Azahara Martinez-Ramirez, Ali Pendle, Isabelle Colas, Alex Jones, Graham Moore and Peter Shaw. The Ph1 Locus Suppresses Cdk2-Type Activity during Premeiosis and Meiosis in Wheat. The Plant Cell. 2012 Jan; 24(1): 152–162.
5. Isabelle Colas, Peter Shaw, Pilar Prieto, Michael Wanous, Wolfgang Spielmeyer, Rohit Mago and Graham Moore. Effective chromosome pairing requires chromatin remodelling at the onset of meiosis. PNAS 2008 105(16): 6075–6080.

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