A pipeline for efficient recombination in wheat

Bread wheat (Triticum aestivum) is a hexaploid cereal with accounts for 20 per cent of the calories and protein consumed by humans and is also an important source of vitamins and micronutrients. It is the largest crop in the UK, grown over 2M hectares, adding over £1.6Bn to the UK economy, with a value almost ten times that for processed wheat-derived products. Despite substantial increases during the green revolution, yields in recent years have plateaued and are now susceptible to decline due to the changes in global weather patterns, in particular increased temperatures. Additionally by 2050, the world's population is expected to rise to 8.9 billion leading to major pressure on resources. As a result of these combined factors there is an urgent requirement to improve wheat varieties through the use of novel approaches based on recent advances in biological knowledge to augment traditional methods of plant breeding. Wheat breeding harnesses the natural process of meiosis, a specialised cell division that leads to the formation of male and female gametes. During meiosis, recombination between the pairs of parental homologous chromosomes generates genetic variation through the formation of genetic crossovers. The progeny that arise are the screened for improvement in desired traits, for instance yield. In wheat, as in most higher organisms the number of crossovers that occur between the parental chromosomes during each meiotic division is low, typically 1-3 and in tends to occur in favoured chromosomal regions. In wheat and some other cereals, this crossover localization is extreme such that most crossovers occur in the distal region near the chromosome ends. As a result large regions of the chromosomes rarely recombine and in effect, these regions become inaccessible to researchers mapping and selecting agronomically important traits. Also it creates the problem of linkage-drag in the recombination-cold chromosome regions where undesirable variation cannot be separated from useful traits. Crossovers arise from the controlled repair of programmed DNA-double strand breaks (DSBs) that are introduced throughout the genome by the SPO11 protein complex. In plants, such as wheat, only around 2.5% of DSBs are repaired as crossovers with the remainder being repaired as non-crossovers. Nevertheless, the DSBs that are not repaired as crossovers are important to allow chromosome pairing and accurate chromosome segregation to form the male and female gametes Our analysis in wheat shows that although DSBs occur throughout the genome they initially form in the distal chromosome regions before appearing in the remaining chromosomal regions. Thus it would appear that most crossovers arise from the earlier forming DSBs, with the later forming DSBs repaired as non-crossovers. This raises the question of whether the repair fate of DSBs can be modified such that crossovers hitherto restricted to distal regions can be redistributed to recombination cold-regions? During the BBSRC sLola upon which this current project is based we used CRISPR-Cas9 gene editing to generate lines in which the A, B and D genomic copies of the SPO11-1 gene have been mutated. We now propose to capitalize on these gene-edited lines to develop an efficient pipeline to create wheat lines in which the level of SPO11-1 activity has been modulated. Specifically we will generate lines in which the number of DSBs and the rate at which they occur is modulated with the aim of modifying the dynamics and repair fate. Additionally, we will use a modified SPO11-1 protein that has been fused to a DNA binding protein-domain to re-target a proportion of DSBs to different chromosome sites with the aim of promoting crossover formation in normally recombination-cold regions. We anticipate this pipeline will provide the basis to maximise the genetic variation that can be accessed by breeders for crop improvement.