18-BTT: Advancing hybrid wheat production through the use of novel pathways for male sterility

The overarching aim of this proposal is to develop novel genetic pathways to induce male sterility in hexaploid (bread) wheat as a route to facilitate production methods for hybrid wheat. The approach is based on preliminary data from studies on rice and maize that have identified pathways which, when perturbed, yield environmentally sterile phenotypes, i.e responsive to photoperiod or temperature. The project will target several pathways, with several targets in the biogenesis pathways that generate abundant small RNAs (21- to 24-nt in length). These two classes of reproductive phased, secondary RNAs (known as 'phasiRNAs') are generated at the critical stages of cell fate setting and during meiosis in grass anther development. Disruption of their biogenesis or encoding loci in both rice and maize leads to conditional male sterility. The proposal exploits these observations to target and disrupt these phasiRNAs in wheat and assess the consequences of this disruption on male sterility. The project will complement work on this pathway by targeting at least one other pathway known to generate a similar phenotype. The PIs' expertise covers small RNAs, plant genomics and targeted mutagenesis, bioinformatics, wheat genetics, cytogenetics and meiotic studies. The proposal is risky but there is preliminary data to suggest all aims are achievable. Success in this area could increase wheat yields by at least 10%, potentially more, representing an extra ~20 million metric tons of wheat in just the EU and US alone, grown with the same inputs and footprint.

Our specific contribution to this project is to generate CRISPR and TILLING mutants. We will transform the spring wheat variety 'Fielder'. For wheat transformation, we will use a pGreen-based MoClo cloneable vector, referred to as pGoldenGreenGate (pGGG), which was developed at JIC. The CRISPR-Cas9 system presently employed at JIC uses the human codon-optimised Cas9 driven by the strong monocot Zea maize ubiquitin promoter. The guide RNAs are driven by the wheat Pol III promoter TaU6. Our strategy is to produce two constructs per target gene with a minimum of two guides per construct usually targeting the first exon in the homologous regions within the three wheat subgenomes.
In the case of the TILLING lines, we have screened the wheat TILLING populations for Tadcl5 mutants, and identified several lines with deleterious mutations in every single copy, both in hexaploid and tetraploid wheat. Additionally, double mutants with all the three genome combinations are available, which means that only one cross will be needed to obtain the triple mutant. Therefore, during the first year of the grant, we could have all the single, double and triple mutants combining the different copies, which will allow us not only to assess the effect of deleting all TaDCL5 copies. In the case of TLLING mutants for the carbon starved anther genes, we will use the same TILLING populations to generate genetic variants in wheat CSA. In hexaploid wheat there are three homologues of the CSA gene in rice, one in every homoeologous group. We have screened the wheat TILLING populations for TaCSA mutants, and identified several mutants with deleterious mutation in TaCSA-A1 and TaCSA-B1 copies in both hexaploid and tetraploid wheat; however, no mutants were available for the TaCSA-D1 copy, the full knock-out in tetraploid wheat (in which the D copy is not present). However, a full knockout in tetraploid wheat can be obtained (in which the D copy is not present) and evaluated for its phenotype.

Currently there are few wheat hybrid cultivars grown, generated either based on chemical hybridisation agents or on cytoplasmic male sterility (CMS) systems. The major limitations of the widespread use of wheat hybrids are seed production and costs. The present proposal assesses an alternative route to generating hybrids, based on disrupting the phasiRNA pathway. The broader impacts of the proposed project include the potential to substantially increase wheat yields with no added land area for production or chemical inputs. The work will advance insights into eukaryotic small RNAs, a field of significant breadth and activity with many fundamental discoveries from the plant kingdom. An important impact is on the training and advancement of post-docs who will be trained broadly in plant small RNA biology and genomics, reproductive biology, crop improvement, and computational methods. Academic collaborations with other labs are integral to the project and will extend the impact of the experimental, computational, and genomics techniques that are an integral part of the project