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Contacts: Andy Phillips and Hai-Chun Jing

Current status

Rothamsted Research initiated a TILLING project in hexaploid wheat at the end of 2003 under the Defra WGIN programme as an underpinning resource for allele discovery and functional genomics, in support of wheat molecular genetics and pre-breeding. The initial aim was to show proof of concept - to demonstrate that TILLING was feasible in wheat: to isolate novel alleles in a small number of candidate genes of interest and take these through to phenotyping. This involved establishing an appropriate TILLING technology platform and also developing mutagenised populations of bread wheat (in collaboration with JIC) and of a diploid wheat, T. monococcum. In support of this programme, we successfully applied to the BBSRC Research Equipment Initiative in 2004 and obtained funds for three Licor DNA Analysers and a Beckman liquid handling robot for automation of the TILLING procedure.

RRes have developed strategies for target identification and homoeologue-specific primer design. An EMS-mutagenised population of ~4,500 lines of cv. Cadenza is stored as M3 seed, and genomic DNA from all M2 lines has been extracted and is available for analysis. The population has been fixed at M6 by colleagues at the Hungarian Academy of Sciences, Martonvásár, and has been successfully screened for mutations in a number of characters. A similar mutagenised population in cv Paragon has been developed at JIC and has also been fixed by SSD to M6. Based on preliminary data (using two target genes across 500 M2 lines), the estimated mutation frequency at any individual nucleotide in the RRes Cadenza population is approximately 0.005%, corresponding to ~200 mutations per kbp within the M2 population. This is a very high mutation density compared with that obtained in diploid species such as Arabidopsis and barley and suggests that even rare, knockout mutations such as those introducing stop codons and splicing errors could be found by TILLING relatively few (1-2,000) individuals. Thus, deep levels of sample pooling that are necessary for efficient TILLING in diploid species are not required, and two-fold pooling is probably optimal.

In addition to the diploid and hexaploid wheat populations, mutagenised populations of tetraploid (durum) wheat (cv. Cham1) are being developed under the EU FP7 'Optiwheat' programme, which aims to improve drought tolerance in this crop. They anticipate that this population will also have high mutation rates and that this may be suitable platform for cereal functional genomics, combining large numbers of novel alleles with relatively low ploidy levels.

RRes now offers a 'hotel' facility for groups to collaborate on isolation of novel alleles in their target wheat genes. TILLING in Brassica crops is being established in association with the international Brassica community.