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Investigating cross species plasticity of meiotic protein complexes to modulate recombination using synthetic biology

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

Co-supervisor: Prof Ed Louis

PhD project title: Investigating cross species plasticity of meiotic protein complexes to modulate recombination using synthetic biology

University of Registration: University of Leicester

Project outline:

Genetic exchange is a key process for genome evolution and is dependent on crossover (CO) formation during meiosis. The frequency and distribution of COs is highly regulated to ensure that each pair of homologous chromosomes receives at least one CO, thus ensuring correct chromosome segregation and normal fertility. CO formation depends on the interaction between chromatin, recombination protein complexes, cell cycle progression and formation of the synaptonemal complex, a meiosis specific proteinaceous structure. CO formation is highly conserved in eukaryotes, and a considerable number of orthologues have been identified across species. However, the frequency and distribution of COs is highly variable between species, suggesting that small changes can lead to large effects. In the majority of higher eukaryotes the frequency and distribution of COs is often restricted. This is problematic for generating novel crop plants with advantageous alleles. Budding yeast on the other hand has proportionally high levels of COs which could be transferred into crop plants.

In this project we aim to transfer known meiotic protein complexes from budding yeast to Arabidopsis and vice versa. We will use a synthetic biology approach to generate ‘biobricks’ that can be easily transferred between organisms with CRISPR/Cas for precise gene targeting. We will initially use the synaptonemal complex proteins HOP1, RED1, MEK1 and ZIP1 from budding yeast and ASY1, ASY3 and ZYP1 from Arabidopsis. We will analyse the products using state-of-the-art fluorescence microscopy (including confocal and super-resolution) as well as molecular marker techniques, fluorescent pollen assays and next generation sequencing.

The specific questions for this project are: 1) Can the synaptonemal complex maintain its normal function in a cross-species genetic background? 2) Can we modulate COs over cross-species barriers to improve crop plants? 3) Which are the key amino acids in the SC proteins that regulate recombination?

Timeline: Miniproject(s) available - Use a bioinformatics approach to identify synaptonemal protein complexes from different strains of yeast and Arabidopsis to analyse co-evolution of particular amino acid substitutions and protein compatibility.

Months 0-1: Use a bioinformatics approach to identify appropriate synaptonemal complex proteins in yeast and Arabidopsis for experimental analysis.

Months 1-12: From selected material generate ‘biobricks’ by cloning synaptonemal complex genes into yeast and Agrobacterium vectors using standard methods and CRISPR/Cas. Obtain mutant lines for synaptonemal complex genes and cross to generate triple/quadruple mutants for complementation of the cross-species genes.

Months 12-18: Transform mutant lines for complementation with cross-species genes.

Months 18-26: Analyse material using cytological techniques and fluorescent pollen assays in Arabidopsis, and NGS in yeast, to characterize the frequency and distribution of recombination events.

Months 26-30: Model data for effect of allelic variation on crossover interference and CO formation.

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BBSRC Strategic Research Priority: Food Security

Techniques that will be undertaken during the project:

We will use CRISPR/Cas and cloning techniques to generate and transfer synthetic biobricks from Arabidopsis to yeast and vice versa. Cytological techniques including fluorescence microscopy, tetrad analysis, QTL analysis (using R), multivariate analysis (using Relative Information Gain and correlation statistics), molecular genetics, Next Generation Sequencing and analysis of the data generated, basic genetics of two different systems, bioinformatics and comparative genomics.

Contact: Dr James Higgins, University of Leicester