Is Rht-a1 a target for the generation of novel green revolution dwarfing genes in wheat?

The primary objective of this short project will be the identification and characterisation of novel Rht-A1 mutations. Currently, we have identified 34 Rht-A1 mutations through screening a mutagenized population of 2000 individuals. A further 2000 individuals are available for screening for mutations using established PCR amplification and high resolution melting analysis techniques. Sequencing and bioinformatics analysis will be used to identify mutations. Sequence analysis and protein modelling approaches will then be used to identify mutations which are likely to block binding to the GA-GID1 complex and hence confer GA-insensitivity. Promising mutations will be introduced into an Rht-A1 prey construct using site-directed mutagenesis. These constructs will subsequently by used in yeast 2-hybrid assays to assess whether they perturb the interaction between Rht-A1 and the GA-GID1 receptor complex. In parallel with these molecular experiments, physiological studies to establish GA-insensitivity in the two available dwarf Rht-A1 alleles will be performed. These characterisation studies will include GA dose response assays to analyse GA-responsive leaf extension.