CORDIS

During a tissue-specific phase of plant development called meiosis, recombination (or crossing-over, CO) drives the exchange of genetic materials and releases genetic diversity by creating new combinations of alleles within and among chromosomes. CO occurs in the small populations of cells that develop into the gametes (eggs and sperm). In plant breeding CO is exploited through the generation of large populations of sexually derived progeny plants from which genetically improved individuals are subsequently selected. However in some economically important crops such as barley and wheat there is an emerging problem: the rate of genetic improvement appears to have plateaued. One hypothesis to explain this is that constraints on the location of CO restricts the release of genetic diversity that is required to drive crop improvement. This is because CO in barley is restricted to the ends of chromosomes, excluding approximately two thirds of the genome from the breeding process.
If genetic gain is reduced and yields continue to plateau, then the long term societal impact will be on the human population that continues to increase while agricultural land and productivity shrinks due to environmental change. Consequently, primary food and feed commodities will become limited. While, at one level, this could result in increased food prices or a scarcity of specific products on supermarket shelves, in poor and subsistence societies it could lead to major issues such as regional famines, conflicts and mass migration.
This project focuses on how we can release genetic diversity from currently inaccessible regions of the genome to improve the rate of genetic gain in crop improvement. We address three critically important questions about CO in barley: Why is it restricted to the telomeric ends of chromosomes? What genes and proteins are the key players in determining this distribution? What are their roles in controlling CO? Answering these questions will allow us to propose then evaluate strategies for modifying the distribution of recombination in barley, and that in a broader context may find application in breeding other major crop plants. A short video of the project explaining the issues we address can be found here: https://drive.google.com/file/d/14_hnleL9zbDD1FjcCydvLnsA3Lu7Pu0C/view

Progress in the first 30 months can be summarised as follows:
• We have generated extensive mutant populations in the cultivar Golden Promise and developed both forward and reverse genetic strategies for identifying mutations in genes that affect meiosis. We have developed a novel screening protocol based on custom exome capture sequencing and identified mutations in many meiotic genes. For forward genetics we have isolated and multiplied a collection of approximately 250 semi-sterile lines of cv. Golden promise and 180 of cv. Optic and are screening these by exome capture sequencing and cytology. We have also extended this sequence based screening approach to look at natural variation in meiotic genes in the wider barley genepool.
• We have made a collection of CRISPR –cas9 single and double mutants and RNAi knockdowns of genes shown in different species to change the distribution or increase the rate of recombination. In many cases the plants appear to be semi-sterile. They are currently being crossed to generate appropriate populations to test the effect on recombination by genetic mapping of F2 families.
• We have developed a reference genome assembly of Golden Promise and reference transcriptome of anthers and meiocytes throughout meiosis as fundamental underpinning resources for the project, and are currently conducting RNA-seq based time course expression analysis (note: the GP ref seq will also be used as part of a collaborative barley pan-genome project). We have initiated a collaboration with Joakim Lundeberg (Karolinska Institute, Sweden) to perform spatially resolved transcriptome analysis using inflorescence sections containing meiocytes tissues to overcome issues associated with the isolation of meiocytes.
• We have used forward genetics to clone genes in semi-sterile lines that affect different steps during meiotic progression. To date we have identified lesions in five genes and are using various approaches to establish and understand their role in meiosis at the same time as progressing with isolating others. A considerable focus is now being put on writing up these results for publication.
• We have developed a novel microproteomics protocol (using single 0.5mm developing anthers) which is exceptionally sensitive and robust, and demonstrates excellent biological and technical reproducibility. We have shown that it compares favourably with time-consuming macroprotomic approaches and have initiated meiotic time courses (similar to RNA-seq) and comparisons between WT proteomes with those from mutants.
• We have are exploring the landscape of recombination in populations using various computational tools and genotypic datasets based on linkage disequilibrium. These statistical methods have highlighted hotspots and coldspots in the barley genome and we are now exploring methods to experimentally test and validate the frequency of recombination in various target regions.
• We have shown that applying various environmental stresses during meiosis can shift patterns of recombination into otherwise recombinationally cold regions of the barley genome. The approach is already being adopted by industrial collaborators in breeding to more efficiently break undesirable linkages.

• Mutation screening-by-sequencing protocols are novel and productive and will generate many novel barley meiotic mutants. Understanding disruptive natural diversity may be a long term productive approach.
• Microproteomics is novel and widely applicable to small plant tissues – will be critical for understanding how disruptive mutations affect the meiotic proteome
• Characterising meiotic genes and their impact on meiosis in a large genome cereal is novel and goes beyond state of the art. We have already shown that some of our novel mutants change patterns of recombination in F2 families and have potential application in breeding.
• CRISPR mutants will assess whether results observed in Arabidopsis transfer to barley.
• Understanding the recombination landscape in different genepools will potentially reveal different evolutionary histories.
• New collaborations in spatially resolved transcriptomics (assuming they are productive) are potentially very powerful and may supersede the likes of laser capture or in situ hybridisation.
• Applying environmental stresses during meiosis are already being used by plant breeders in parallel projects.