Initially we focused on identifying mutations in the barley genome that affect meiosis. We used a potent mutagen called EMS to develop a large mutant population of GP. We then searched for lines that were semi-fertile – which is a hallmark of meiotic mutants – and identified several hundred. To screen these at the sequence level we developed a novel sequence-based using target enrichment sequencing. By characterising ALL of the semi-fertile lines we identified a large number of lesions in many of our targeted genes. We also adopted an approach called a ‘suppressor screen’ where we attempt to identify semi-fertile lines that have their fertility restored due to mutations in a different gene identifying a small number of suppressor lines. Finally used Genome Editing to produce knockout alleles of meiotic genes that had been shown previously to affect CO. We prioritised a few of the most promising mutants for detailed characterisation.
We used and high resolution immuno-cytology to investigate where, when and what the impact of the mutations we had identified were on meiosis. We also constructed populations segregating for mutant alleles and used genetic segregation analysis to compare the effect of mutant vs. wild type versions of the identified genes. We identified mutants that changed CO frequency and location. We found that some of the genes we identified had been identified previously in different species (e.g. MLH3). However, in barley we observed either subtly or radically different effects. Other mutated genes were novel. One, STICKY TELOMERES 1, is an E3 ubiquitin Ligase where mutants increased CO by >2.5 fold. In addition two lines identified from the suppressors screen restored recombination from <50% to well-above wild type levels. These were mutants in genes called FANCM and RecQL4, previously shown to restore recombination in Arabidopsis.
To discover novel proteins we also looked at the protein repertoire of meiotic cells. We developed a micro-proteomics workflow to profile the proteome individual meiotic phase barley anthers (the structure containing the meiocytes,) highlighting over 300 that changed in abundance during meiotic progression. In parallel we developed a meiotic anther RNA transcriptome using techniques that allow us to survey almost all of the RNA molecules in a cell. We identified many differentially expressed meiotic genes and assembled them in modules that showed different expression patterns during meiotic progression. These experiments have led us to focus on a family of ARGONAUTE proteins, highly specialized proteins that bind small RNAs and coordinate downstream gene-silencing events, that exhibit contrasting patterns of expression.
We studied the natural patterns of CO in barley using a combination of genetic approaches that examine CO at high resolution in natural bi- and multi-parent populations. The results show that there are ‘hotspots’ of recombination in the barley genome, just as there is a massive ‘cold spot’ across the centromeric regions. The hotspots tend to be conserved across families. We explored why the ‘cold’ regions are recombinationally inert. We observed few ancestral genomic patterns (called haplotypes), peppered with variants most likely introduced by non-crossing over or gene conversion events. To extend this work we developed a novel method for assessing CO using mutagenesis and high throughput sequencing reducing the time and expense of genetic analyses
We are now focused on exploring how our discoveries can be used in breeding.
