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Enhancing meiotic recombination in wheat by modulating RECQ helicases

Primary Supervisor: Dr James Higgins, Department of Genetics and Genome Biology

Secondary supervisor: Prof Ed Louis

PhD project title: Enhancing meiotic recombination in wheat by modulating RECQ helicases

University of Registration: University of Leicester

Project outline:

Wheat accounts for 20% of the calories and protein consumed by humans and is the largest crop in the UK, grown over 2M hectares, adding over £1.6Bn to the UK economy (https://www.wheatinitiative.org). Despite substantial yield increases during the green revolution, yields have now plateaued and are susceptible to decline due to extreme weather patterns. Wheat breeding is a numbers game; the more crosses generated, the greater the chance the breeder has of generating useful combinations of genes (alleles) that lead to a desired phenotype. However, breeding is dependent on the frequency and distribution of crossovers (CO) that are few in number (1-3 per chromosome pair) and skewed towards the chromosome ends, so even with a large number of crosses, desired combinations may not be attained. CO initiation sites are not limited in the genome by location or number, but only ~2% mature into actual COs. Therefore, new approaches are needed to increase COs in cereal crops and improve the efficiency of breeding programmes.

In humans, mutations in the RECQ genes, Bloom’s syndrome helicase (BLM) and Werner’s helicase (WRN) are associated with premature ageing and early onset of cancer1. RECQs are conserved throughout eukaryotes and repair DNA by homologous recombination. In Arabidopsis thaliana, the duplicated RECQ4a/RECQ4b genes are the BLM orthologues2 and function redundantly as anti-recombinases during meiosis and by mutating these genes increased COs ~6.2-fold3. A WRN ortholog is not present in Arabidopsis, but we have recently identified one in wheat (RECQ7)4, as well as the BLM ortholog, RECQ4. We have determined that RECQ7 is a pro-CO factor, and RECQ4 is an anti-CO factor, so the two proteins may act antagonistically in processing DNA recombination intermediates down different repair pathways. We have generated knockout mutants of RECQ4 as well as wheat RECQ7 overexpression lines tagged with fluorescent proteins. The mutants and overexpressing lines require analysis as well as determining the mechanism behind altered CO patterns. We will use state-of-the-art super-resolution fluorescence microscopy in conjunction with immunolocalisation and a panel of antibodies developed in the lab that target specific proteins in the CO pathway. Using this approach we will be able to determine the spatio-temporal dynamics of recombination protein loading on meiotic chromosomes. We will also utilise a genomic approach to monitor CO frequency and distribution either through molecular markers or by whole genome skim-sequencing. As part of the training and to gain a fundamental insight into the function of the RECQ helicases, budding yeast will be employed as a model organism to determine the effect on recombination of RECQ4 and RECQ7 across two different kingdoms. Budding yeast possesses a RECQ4 orthlog (SGS1), but not a RECQ7 ortholog, similarly to Arabidopsis, which may reflect genome size, chromosome structure or chromatin environment. There are three outstanding questions that will be investigated in this PhD project:

Q1. How does RECQ7 function as a pro-recombinase in homologous DNA repair in wheat?

Q2. Does RECQ4 act as an anti-recombinase during meiosis in wheat?

Q3. Can we determine an antagonistic mechanism of action between RECQ4 and RECQ7 using both wheat and budding yeast?

The project will be directly translated into crops as well as providing a fundamental knowledge on homologous DNA repair mechanisms in eukaryotes. You will be trained in all modern techniques in an active research lab, with the opportunity for training in external collaborative labs.

References:

  1. Croteau et al., 2014, annualreviews.org. 2014; 2. Hartung et al., Proc Natl Acad Sci USA, vol. 104, no. 47, pp. 18836–18841, 2007; 3. Seguéla-Arnaud et al., Proc Natl Acad Sci USA, vol. 112, no. 15, pp. 4713–4718. 2015; 4. Gardiner et al., Genome Biol, vol. 20, no. 1, p. 69. 2019.

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Plant and Crop Science

Techniques that will be undertaken during the project:

  • Dissecting microscopy
  • Fluorescence microscopy (widefield and confocal)
  • DNA sequencing
  • PCR, cloning
  • Crossing wheat genotypes
  • Yeast tetrad analysis

Contact: Dr James Higgins, University of Leicester