Go to the TILLING website for further information and information on submitting genes for mutant analysis in our collections.

A platform for reverse genetics in barley

The past decade has seen a dramatic increase in the number of gene sequences that have become available for various species including barley. SCRI was heavily involved in developing and sequencing Expressed Sequence Tag (EST) libraries as part of the Investigating Gene Function (IGF) programme with funding from the Scottish Government and BBSRC. This sequence data identifies genes which are expressed in a given plant but provides little information about what the function of the gene is. The SCRI Genome Biology group have a number of approaches to identifying gene function and in particular identifying genes with functions which relate to important traits for barley breeders, maltsters and brewers.

One approach is to disrupt the function of the gene and analyse how this alters the properties or 'phenotype' of the plant. This is the basis of the method known as TILLING.

Development of a structured mutant population

We have used Ethylene Methyl Sulphonate (EMS) to generate a population of approximately 22,000 lines. EMS introduces methyl groups into the DNA which leads to errors during DNA replication and thus introduces mutations. Typically this means that some C nucleotides within a sequence are replaced with T residues and also G residues with A residues. Within our population this occurs at random sites at a frequency of approximately 1-5 changes in every 100000 nucleotides. The process by which a mutant population and then mutants are identified in specific genes is shown in Fig. 1 (below). We have recently bought three LiCOR sequencing systems which allow the detection of mutants using the Cel 1 digestion method outlined in Fig. 1. The results of one of these gels are shown in Fig. 2.

We are currently funded by BBSRC and the Scottish Government to develop this protocol to provide a service of mutant isolation for the research and industrial community in the UK. We are currently collaborating with many laboratories in the UK and across the EU to identify mutations in candidate genes which may be involved in sprouting (germination), nutrient partitioning, flowering time/vernalisation, pathogen resistance and malting quality.

Figure 1. Generation of a barley mutant population with EMS and its use for TILLING.

Figure 2. LiCOR sequencing gel used to identify mutants in a barley gene CTS2. Showing truncated “mutant” fragments at 320nt and 340nt in the 700nm channel and complementary fragments at 650nt and 630nt. Mapping of the mutation sites within the amplified CTS2 gene.