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

Lead Research Organisation: John Innes Centre
Department Name: Metabolic Biology


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.

Technical Summary

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 ( 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.

Planned Impact

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.
Description We have established a series of populations of plants (both models and crops) for use in scientific research and crop breeding programmes. The populations cover brassicas (B. rapa, B. oleracea, B. napus, Capsella rubella), legumes (Lotus japonicus, Medicago truncatula) and cereal species (wheat and oats, with access to rice from China). DNA from each of the plants in a population is used in a process known as TILLING, a reverse genetics method. Using this process we find variants of a gene in which any researcher is interested, so they can test the function of the gene. Useful variants can then be deployed in research or breeding programmes for crop improvement. In this programme since its inception, we have carried out TILLING on about 400 gene sequences (about a quarter in this particular project period).

We have established an international TILLING platform, RevGenUK, that is open and free of intellectual property constraints, for use by researchers in academia or industry. Since it started operating, RevGenUK has found variants for more than 100 clients from across14 countries.

In addition to the populations established for conventional TILLING an entirely new method for capturing variants in mutagenized populations was developed as one of the objectives. We took approx. 1000 plants of one particular species, Brassica rapa, and developed methods to obtain the full gene coding sequence (exome) for EACH line using next generation sequencing. This was successful and in so doing we have captured information for about 1.3 MILLION MUTATIONS. This data has been made publically available online at our website ( and elsewhere. This allows anyone to look for any gene of interest and instantly find variants to use for research or breeding purposes to improve crop traits, such as yield or nutritional value.

We have also made improvements to the methods that should make the platform more efficient and cost effective in the future.
Exploitation Route RevGenUK is now an established UK platform for reverse genetics in plants that offers its services internationally. It has been widely publicised (via presentations, posters and flyers) to researchers through scientific meetings, such as the UK Brassica Research Community, through Defra Genetic Improvement Networks, through annual scientific conferences both international, such as the Plant and Animal Genome meeting in California, and at UK conferences, such as the Society for Experimental Biology. The platform has been incorporated into the John Innes Centre's services that are offered by the Norwich Research Park's Virtual Technology Centre (

More plant species have been added to the platform for use in TILLING, for example rice (Oryza sativa Japonica) that was developed with the Institute of Botany, Beijing, and oat (Avena strigosa). Grass pea (Lathyrus sativus) is under development in house currently.

Data from this platform are available over the web and can be used by researchers to identify variants that will help them understand how plants work, and more especially be used by plant breeders, who can incorporate them into their programmes for crop improvement. The methodology developed in the project can be used by other researchers and applied to their plant species of interest.
Sectors Agriculture, Food and Drink

Description RevGenUK (RGUK) evolved from research into a single model legume species and is a reverse genetics TILLING platform for obtaining plant mutants in cereals, brassicas and legumes. These mutants can be used for academic research or commercial plant breeding purposes. We have carried out more than 400 TILLING operations during the life of the platform (ca. 100 in this award) that have been fed into research programmes and several breeding programmes across the globe with a view to improving crop plants. As research tools, the mutants have been used to understand how plants work and the findings published in peer-reviewed academic journals, those from the PI on this award have been recorded accordingly. Others researchers have used the mutants for their own research and the basis of grant applications and project with other academics and industrial partners. Directly from RGUK results, a LOLA initiative has been proposed (Brassica Rapeseed And Vegetable Optimisation, BRAVO) with the goal to elucidate, validate and exploit the interlinked processes that determine trade-offs between developmental traits. This knowledge will provide the foundation for reliable, high-marketable yields and the robust production of high-quality seed in oilseed and vegetable Brassicas and provides the opportunity for interactions between academics and industry.
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Economic

Title Brassica rapa mutation browser 
Description This database (web browser) allows one to browse Brassica rapa genes and look for mutations in particular regions. You can then retrieve and display positional and sequence information on each mutation 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This is one of the first examples of comprehensive mutation data obtained by exome capture and genomic resequencing in plants. 
Title RevGenUK TILLING database 
Description A database of mutations in selected species of plants derived either from TILLING or re-sequencing whole genomes 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This database allows researchers to find mutations in their gene of choice in a number of species of plants for plant functional genomics research