RevGenUK, the next generation: establishing a TILLING boutique for a UK-based reverse genetics community resource

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The genome sequences of many plants have been established, especially for models such as Arabidopsis thaliana for dicots, Lotus japonicus for legumes and Brassica rapa for brassicas. Sequencing the major UK cereals is also underway as exemplified by barley. To uncover the function of the thousands of genes that will be discovered by these initiatives will be a major challenge. One approach to address this challenge is the use of reverse genetics. Reverse genetics uses genome sequence to permit the recovery of mutants in target genes for use in functional genomics. This is achieved in many plants using TILLING, a process whereby individual nucleotide changes in a sequence can be identified using a process of DNA heteroduplex formation and enzymatic cleavage at mismatch sites. This is a non-GM method that generates an allelic series of mutants including nulls, and also permits the mining of natural variation. We have assembled populations for TILLING in L. japonicus, Medicago truncatula and B. rapa and have established a TILLING service (http://revgenuk.jic.ac.uk) for the research community. In this project we will extend this platform to barley and oats, and provide a bespoke service for academics and industry. Mutation detection has been carried out until now by capillary sequencing, but we will augment the platform by developing TILLING by re-sequencing using gene capture and NextGen (Illumina) methodologies. This will encompass the development of appropriate pooling and library construction strategies coupled to a robust bioinformatics pipeline and an update of our existing database to include mutation information on all our species. Under the umbrella of the single technology platform, therefore, we will create a one-stop-TILLING-shop and a unique UK resource for mutation discovery and functional genomics for the benefit of the research community.

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