The role of E3 ligases on the modulation of recombination in cereals

Supervisors: Robbie Waugh, Isabelle Colas

Project Description

Overview/Background

In barley and wheat substantial proportions of the chromosomes are inherited together as a large linkage block, preventing the generation of novel combinations of alleles that 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.

Aim/Scope

Meiotic recombination is highly controlled to ensure the species genome integrity.  Recent findings have described so-called anti-crossover factors such as FANCM, RECQ4 or FIGL1, that once downregulated, increased meiotic recombination (in Arabidopsis). Utilizing the barley mutant collection held at the James Hutton, we mapped a spontaneous mutation, des4, on chromosome 7H and identified a novel gene. Despite its 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 of each chromosome 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 STTP1 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 detail the mode of action of STTP1 in controlling crossovers in barley and wheat.

Methods/Approach/outcomes – 0-18 months

  1. Cytological analysis such as time course with EDU and immuno-cytology with antibodies against a range of meiotic proteins will be used to deepen our understanding of the mode of action for STTP1, in particular around the telomeric regions.
  2. There is some evidence in the literature for interactions between STTP1 and des11 mutants.Large F2 mapping populations are available to rapidly map and potentially clone des11 which may help explore the mode of action of STTP1 through the production of double mutant lines. The student will make use of a new 50K iSelect genotyping chip to simplify this analysis along with the recently characterized barley genome sequence.
  3. As there is currently only a single mutant allele of STTP1 we want to explore whether an allelic series of potentially less deleterious alleles have a sterility and meiotic phenotype.We will generate / discover additional mutants by a combination of TILLING and CRISPR-Cas induced mutagenesis.
  4. Populations will be developed for new mutants from 3. for mechanistic and genetic studies
  5. sttp1 mutants in barley display a large number of interlocks that could potentially cause the increased crossovers.  We will identify and order similar mutations in wheat from the TILLING populations held at the John Innes Centre and test for semi-fertility.  We will initiate cytological analyses for the presence of interlocks, telomere fusion and abnormal synapsis. Crosses (including to commercial lines with the 1R/1B rye translocation) will be made to look at the impact on genetic segregation and the potential for increased recombination between homologous and non-homologous chromosomes.

Methods/Approach/outcomes – 18-42 months (+6 months for writing)

Since E3 Ubiquitin ligase (such as RNF20) have been shown to alter chromatin conformation by altering H2B ubiquitination, a detailed analysis of chromatin states will be established by cytology and the development of ChIP-sequencing for specific histone modifications in relation to DSBs sites, starting in year 2 of this PhD.  If time allows, the student could also develop a co-immunoprecipitation method with STTP1 antibodies to explore the protein network associated with this novel gene. This would potentially lead to the identification of STTP1 containing protein complexes, and new genes involved in the ubiquitination pathway of early meiosis in barley.

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. Phillips, D., Jenkins, G., Macaulay, M., Nibau, C., Wnetrzak, J., Fallding, D., Colas, I., Oakey, H., Waugh, R., & Ramsay, L. (2015). The effect of temperature on the male and female recombination landscape of barley. New Phytologist, 208(2):421-9.
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