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

In an ever changing climate, we are constantly trying to improve crops to achieve more sustainable agricultural practices. To do this, we need to understand in great detail how plants grow and work. Genetics is a powerful tool for helping us to obtain such an understanding. Using it we are able to analyse the whole genetic make-up (the genome) of plants to discover all the genes required for them to develop and operate correctly. We can use this information to study variants that have a defect in a particular gene (or genes) in which we are interested. This helps us to understand how the gene works when it is operating normally and its role in the plant. Many species of plant have had their whole genome sequenced already, but there are still thousands of their genes whose role in the plant is not understood. To obtain plants that bear a defective gene we can treat seeds with a chemical or radiation that damages the DNA coding for that gene. The offspring from these treated seeds will bear a large number of defects in their genomes. By producing a large population of offspring we can make every gene in the genome bear a defect. The problem then is to find the plant in the population that has a defect in the gene in which you are interested. We have developed methods that can sort out defective genes of interest and find the plants that contain them, so we can find out the role of the gene. This whole process is known as reverse genetics. No one chemical or physical means can induce all the defects we need to study particular genes so we need to use several different methods. In a previous project we set up a resource for the plant science research community so that they can discover the function of their particular genes of interest. We did this for legumes and brassicas (treated with chemicals) that contain plants bearing different forms of defective genes, but now we would like to do it for major UK cereal crops to help both scientists and plant breeders. We have developed special methods based on high throughput machines (sequencers) that can detect the defects when compared to the normal gene. Now we wish to develop methods that will do this in a more efficient and cost-effective manner. Scientists can then send us information about the gene of interest, for example, a gene from oats or barley, and we can then look for defects in their specific gene in our populations of thousands of plants. We then send them seeds from the plant that they can grow to study the action of the defective gene in that plant. All the information that we gather about our plants and their thousand upon thousands of genes will be stored in a computer database that we have constructed especially for this project, although it will be written in such a way that others can use it as well. It will also be available to use on the worldwide web so that a scientist anywhere in the UK or the World can come and browse to see if the database contains information about their gene of interest. The reason for wanting to do this is to improve the ability of crop plants to grow in different environments, especially adverse ones, to improve the quality of our food, and to help the farmer work in a sustainable way using less added fertiliser and fewer herbicides and pesticides.

The genome sequences of many plants have been established, especially models such as Arabidopsis thaliana, Lotus japonicus and Brachypodium distachyon. The major UK cereals are also underway, with barley anticipated in 2012. To uncover the function of the thousands of genes that will be discovered by these initiatives will be a major challenge. 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 non-GM method which generates an allelic series of mutants including nulls, albeit the latter at a low frequency, and also permits the mining of natural variation. We have previously assembled or are in the process of assembling populations for TILLING in L. japonicus, Medicago truncatula and Brassica rapa and have established a TILLING service (http://revgenuk.jic.ac.uk) for the research community. We wish to extend this platform to a UK cereal model and cereal crops, 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-sequencing using 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 the six 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.

Who will benefit from this research? 1. Agro-industry including biotechnologists and plant breeders with known plant gene targets seeking to improve all aspects of plant growth, crop yield, development, resistances to stress and product quality. 2. Agricultural community and advisors. 3. Postdoctoral researchers. 4. Public. How will they benefit from this research? 1. Agro-industry will be able to access directly RevGenUK's platform to obtain mutants and receive information that underpins rational approaches to improve plants for food, fuel and feedstocks, improve forage quality, engineer nitrogen fixation into non-legumes, and discover new tools for metabolic engineering and stress resistance. In the short term, we will be able to identify mutations for specific target genes for improving in legumes and brassicas primarily to undertake functional analyses. In the medium term this will be extended to the accepted model and UK crop cereals, but in the long term it will provide material to use in breeding programmes. 2. The agricultural community will benefit in the longer term from sustainable crop improvements enabled by our community resource. 3. The postdoctoral researchers will receive: help with career progression; advanced training in plant integrative biology including 'at the bench' and 'informatics' approaches; courses on professional skills including IP, management and leadership; the opportunity to liaise with researchers worldwide. They will also be linked with related international reverse genetics activities at JIC on sweet sorghum and rice, through our China Partnering Awards and on cassava and grass pea through collaborations with India. 4. Our research findings relate to issues of public/consumer interest including food quality, sustainable crop production and genetic modification. They will underpin a better understanding of gene function and all aspect of crop modification by plant genetics. In the medium to long term advances made by the platform customers will lead to improved food quality, plant yield and resistances to stress, and the application of plant materials to biofuels.