Investigating the role of the meiotic chromosome axes in mediating crossover designation in bread wheat

Bread wheat (Triticum aestivum) 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 hectares1, adding over £1.6Bn to the UK economy, with a value almost ten times that for processed wheat-derived products1. Despite substantial increases during the green revolution, yields have plateaued and are now susceptible to decline due to extreme weather patterns. By 2050, the world's population is expected to rise to 8.9 billion leading to considerable pressure on resources, such as water availability, affordable fuel and food2. Therefore, it is essential to assist plant breeding by providing biological expertise to generate climate tolerant, high yielding varieties, which will benefit both the economy and society whilst contributing to food security in the UK.

In bread wheat, important agronomical traits are encoded in the DNA of forty-two diploid chromosomes. Traditionally, plant breeders cross high yielding parental lines and then select progeny in the next generation with desirable attributes. However, the process of 'gene-shuffling' in wheat is non-random and skewed towards the ends of the chromosomes. Therefore, desirable traits are often transmitted with undesirable traits. To break this linkage requires a novel distribution of genetic crossovers (COs) which is unlikely to occur by conventional means. Studies from the model plant Arabidopsis, as well as cereals such as rice and barley have shown that it is possible to modulate the frequency and distribution of COs by altering the expression of particular genes or inducing a temperature heat shock3. In both Arabidopsis and barley the pachytene checkpoint 2 (PCH2) protein is required to impose the normal distribution of COs and in its absence, COs are detected in regions that rarely or never receive any COs4. It is therefore a suitable target for investigation in bread wheat. In budding yeast, PCH2 interacts with the meiotic chromosome axes to regulate the frequency and distribution of COs. However, the chromosome axes in bread wheat are required to prevent homeologous recombination as well as promote the normal frequency of COs. Therefore, we will need to determine the role of PCH2 in mediating the chromosome axes to maintain homologous COs. The initial step of the project will be to clone the PCH2 homeologs from bread wheat and then quantitatively analyse the expression of these genes. A complementary approach will utilise fluorescence in situ hybridization to confirm the copy number of these genes. This information will be used to generate gene specific primers for identifying TILLING mutants from the population at the John Innes centre in collaboration with Cristobal Uauy. The TILLING lines are directly useful for plant breeders as they are not genetically modified, they do not have associated intellectual property and it is possible to generate hypomorphs based on the type and frequency of mutation. Whilst identifying TILLING mutants, an antibody will be generated to the wheat PCH2 protein by cloning a suitable region and expressing it in E. coli, before purifying the protein. This will be used at a later stage in the immunocytological analysis of PCH2 function. The second step will be to cross the single mutant lines to make double and triple knockouts, as well as crossing with the elite germplasm. The crossing will take place at the KWS plant breeding site in Cambridge, UK, supervised by Dr. Ed Byrne and Dr. Nicola Kettles. Mutations in the progeny from these crosses will be confirmed by PCR and then analysed cytologically, using chromosome spreads to quantify the frequency and distribution of chiasmata, the cytological sites of COs and by mmunolocalisation, using antibodies raised against a panel of meiotic proteins in Arabidopsis (that work in barley3) to monitor the different steps of recombination and determine how PCH2 functions