Performance Story: Development of fully cleistogamous wheat and associated markers
Fusarium head blight (FHB) is a devastating disease impacting wheat production in Canada and around the world. Cleistogamous flowers do not open and pollen-containing anthers remain inside the flower. In barley (Hordeum vulgare) and wheat (Triticum aestivum) this reduces the ability of fusarium to initiate infection during flowering. Cleistogamous barley is caused by a mutation in the HvCly1 gene within a microRNA (miRNA) cleavage site that controls gene function. During normal flower development, a miRNA binds to this site and triggers the degradation of HvCly1 mRNA thus turning off the gene. This triggers normal anther extrusion. In Cleistogamous barley the binding site has been changed, cleavage does not occur, anther extrusion is not triggered and FHB incidence is reduced.
Wheat is a hexaploid species with a Cly1 gene in the A, B and D genomes. Combining mutations in the miRNA cleavage site of all three wheat Cly1 (TaCly1) genes should produce cleistogamous wheat flowers. The objective of this project was to identify null mutants (complete loss of gene function) and also mutations in the miRNA cleavage site for TaCly1 A, B and D using an approach called allele mining, which identifies and exploits natural genetic variation and avoids the regulatory hurdles of genetic modification. Perfect markers were generated for all variation (alleles) to facilitate combining alleles to test for the cleistogamous condition and enable use of this material in breeding programs.
During this project 837 landraces and 863 lines exposed to the mutagen ethyl methanesulfonate were screened using two strategies 1) amplification of DNA fragments to detect large changes (inserts or deletions) in the genes 2) DNA sequencing to detect small mutations. TaCly1 null alleles for all three genomes were identified and perfect markers facilitated the generation of a line combining all three alleles. This line had normal anther extrusion. Mutations were identified in the miRNA binding region of TaCly1 in the A, B and D genomes. An assay to test the impact of these mutations on messenger RNA (mRNA) cleavage determined that the TaCly1 A variant blocked cleavage while the TaCly1 B and D variants did not. Perfect markers for all three variants were developed, however, a single line containing all three variants was not generated because the cleavage tests indicated that the TaCly1 B and D would not contribute to cleistogamous flowers. Lines homozygous for the TaCly1 A variant had normal anther extrusion even when combined with the TaCly1 B and D null alleles. We believe that anther extrusion was unchanged because the degradation of TaCly1 B and D mRNA will trigger degradation of the TaCly1 A variant even though its miRNA binding site is not functional. The null alleles identified in this study produced mRNA and still triggered degradation the TaCly1 A variant.
This project demonstrated both the power and limitations of an allele mining strategy for crop improvement. The identification of null alleles was straightforward, however, the identification of mutations that specifically block mRNA cleavage was only possible in one of the three genomes. The allele mining approached is more likely to be successful when a null mutation is required. When a more specific mutation is required, such as modification of an mRNA binding site, the probability of success should be carefully evaluated. Allele mining could eventually identify TaCly1 B and D cleavage variants, however, is it unclear how long that would take. It would also be worthwhile to confirm that the desired mutations would actually produce the desired effect using CRISPR gene editing before investing in an allele mining strategy. It is very likely that combining TaCLy1 A, B and D variants that are no longer cleavage my miRNA would produce cleistogamous wheat; however, this is based on barley data.
In summary, this project was successful in identifying both null and miRNA cleavage site variants in the A, B and D genomes but this material did not produce cleistogamous wheat flowers.