Provision of TILLING resources and platforms in wheat.

Lead Research Organisation: Rothamsted Research
Department Name: Plant Biology & Crop Science

Abstract

Wheat is a vital food crop, planted on over 200 million hectares of land globally and providing about 20% of the calories that we consume. Yields of wheat increased dramatically in the last century, particularly in the developing world, due to a combination of plant breeding and increased use of fertilisers and pesticides. More recently, yield improvements have slowed and in many countries may be levelling off. A host of factors such as population growth, change in dietary patterns, climate change and use of land for biofuels have highlighted the importance of food security and demonstrate the need for continued improvement in wheat yields and in the efficiency with which crops use inputs such as water and fertilizer. This comes at a time of rapid advances in our understanding of plants that will greatly assist in knowledge-based crop breeding. Work on so-called 'model species' is identifying genes and pathways that are important in plant growth and productivity. At the same time, new technologies are being applied to biological questions, resulting in an explosion of information on the genetic makeup of plants in general, and crops in particular. This will identify 'candidate genes' in wheat that may play significant roles in crop yield and quality. In order to test the importance of these genes or to manipulate their activity to improve crop performance, we need the ability to routinely modify gene function in wheat. While this can be achieved through genetic transformation, transgenic plants have yet to gain consumer acceptance in Europe. As an alternative, we can use natural or induced variation in these candidate genes to investigate their function. However, the wheat genome is very complex (containing sixteen billion base pairs of DNA) and until recently identifying small differences in specific genes was an impossible task. Recently, plant scientists developed a method called TILLING which makes the mutation discovery process more feasible. This technique allows researchers to search within a large collection of plants for those carrying mutations in a single gene of interest. Those individuals identified can then be compared to assess the role of the gene in determining plant performance, and in crop species those with improved properties can be entered into breeding programmes to produce new varieties. TILLING is potentially a very attractive approach to accessing genetic variation in crop species, but it has several limitations. Although TILLING is efficient, the existing methods are quite complex and require expensive equipment. In addition, certain crops such as wheat are not ideally suited for this technique due to inherent complexities in their DNA. Equally important, scientists need access to wheat TILLING lines to be able to screen them, but not all of the existing resources are publicly accessible or in a readily usable form. In this proposal we will organize wheat TILLING as a public and open access resource. We already possess suitable collections of both bread and pasta wheat lines containing natural and induced mutations, and we will make these readily available by preparing large quantities of DNA and seeds for each line, and provide these free to UK researchers. We have developed a low-cost TILLING method that should enable all interested scientists and plant breeders to perform this technique in wheat. As part of this project, we will offer practical training courses on these methods to reduce further the barriers to wheat TILLING. We also aim to establish new methods for identifying mutations by high-throughput sequencing. We are convinced that by making this technology more accessible to researchers we will advance our understanding of gene function in wheat. This is the first step in modifying these genes to help wheat breeders produce new varieties with improved yield, better nutritional properties and better resource use efficiency for the benefit of consumers, farmers and the environment.

Technical Summary

We are entering a new era of crop genetics: work on model species is identifying genes that have major effects on important traits. At the same time, new technology is speeding the sequencing of crop genomes - several have already been completed and a BBSRC project on wheat will generate 5x sequence coverage. These advances provide a major opportunity to modify crop performance through manipulation of candidate genes. However, access to technologies for gene modification in wheat remains limited, especially as transgenic crops have not gained acceptance in Europe. An alternative approach is to identify sequence variation in wheat genes, either in germplasm collections containing natural variation or in populations bearing induced mutations. The TILLING technique allows access to such sequence diversity but is expensive for individual groups and technically challenging, particularly in species such as wheat with very large, polyploid genomes. In this proposal we aim to provide wider access to TILLING technology and sequence variation in wheat for the benefit of the wheat genetics and breeding communities. We have previously generated mutagenised populations of bread and pasta wheat and in this project we will generate large scale DNA samples and grain stocks to distribute to users. We will also provide a set of wheat germplasm containing natural sequence variation. We have developed a simple, inexpensive TILLING assay that will allow users to screen our populations for variation in their candidate genes and we will provide training in this low-tech screening method. The increasing throughput and falling costs of DNA sequencing also suggest a more direct approach to TILLING. We will explore alternative strategies of TILLING-by-sequencing in order to assess the feasibility of the methods and the throughput that can be achieved, with the longer term aim of developing protocols that can be used by existing service providers to provide custom TILLING services in wheat.

Planned Impact

Within the last 25 years, there has been an expansion of knowledge of the genes controlling important biological pathways and their effects in model species. A major challenge, now beginning to be realised, is to move these advances into crop species. One of the biggest obstacles to achieve this translation is the limited technology available for gene modification, a necessary first step to understand gene function. It is with this knowledge that genes can then be modified to address specific aims such as increased resource use efficiency, optimized yield parameters, and enhanced disease resistance, among others. We aim to bridge this gap by providing the research and breeding communities with the resources to achieve gene modification in wheat through non-transgenic means. We also anticipate that this resource and its training component will enable and encourage investigators working in other species to expand and 'translate' their work into this important crop species. We expect multiple beneficiaries of the proposed work, including the academic sector (detailed in academic beneficiaries), the commercial private sector in the form of wheat breeding companies, public sector policy makers, the UK environment and the wider public in general. The development of this resource would allow researchers and breeders directly to address three of the BBSRC Strategic priorities: Bioenergy, Living with Environmental Change and Crop Science (Food Security), thereby addressing policy makers' strategic goals. We are using TILLING within our own laboratories to develop alleles that extend grain filling period, improve harvest index, reduce anti-nutrients such as phytate, increase cell wall digestibility for cellulosic ethanol and increase resistant starch (amylose). These are just a few examples of how we can now modify specific genes in wheat controlling important biological aspects and that directly address BBSRC and government's strategic priorities. This project will make these TILLING resources available to a much wider group of users. Once researchers and breeders have used TILLING and other techniques to understand the genes controlling specific traits (in either wheat or a model species), TILLING also provides a very efficient way to modify these gene and quickly transfer this knowledge to the field. Therefore the resource generated in this proposal should inform on strategies to develop enhanced wheat varieties and provide the means to achieve this. Wheat breeding companies are the key conduit to channel this research into commercial wheat varieties. We have included two UK commercial breeders in our advisory group and the breeding community has provided strong support for this proposal. This strong collaboration with industrial partners ensures that novel alleles generated by TILLING will be translated into commercial wheat varieties, ensuring that the potential impacts of this work are quickly transferred to society. This will directly benefit farmers by providing wheat varieties with enhanced characteristics such as improved resource use efficiency and reduced wastage. These benefits will have a broad impact in social well being and in the UK environment as wheat occupies over 2 million hectares and has a production value of over £2.2 billion (2008).
 
Description We developed a resource of 2000 DNA samples from mutant lines of wheat and 2000 corresponding grain samples. These were used to identify novel mutations in key wheat genes for crop improvement.
We also assessed the feasibility of using exon capture technology followed by next-generation sequencing to identify mutations in coding regions of genes within the EMS population. This was shown to be very efficient and was used to develop one part of the sLOLA in Wheat Genomics (BB/J003913/1)
Exploitation Route The resources from this project have been used by many visitors and collaborators to identify mutations in their target genes. We also contributed to a new project that aims to use exome enrichment and next-generation sequencing to provide data on all coding mutations in 1500 lines of this population.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

 
Description The EMS-mutagenized lines of wheat further developed in this project were used in a follow-up project in which 1200 lines were characterised by exon-capture and sequencing. These lines now form a valuable resource for wheat biologists and breeders: so far, 270 wheat EMS lines have been requested by commercial wheat breeders for the development of new varieties, and licenses for all 1200 lines have been purchased by a major biotech/breeding company. 2019 update: The TILLING lines generated in this project are help in the stock centre at JIC. To date, there have been a total of 250 requests for seeds of specific lines, comprising 6190 seed lots. These include 130 requests for commercial use. This resource is thus a major contribution to academic and commercial work on wheat ffunctional genomics, hypothesis testing and breeding in bread wheat.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Energy,Manufacturing, including Industrial Biotechology
Impact Types Cultural,Economic

 
Description BBSRC Follow-on Fund: Low viscosity wheat for improved properties for fermentation and animal feed
Amount £144,811 (GBP)
Funding ID BB/K010824/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2013 
End 08/2014
 
Description BBSRC sLOLA: Triticeae Genomics for Sustainable Agriculture
Amount £36,822 (GBP)
Funding ID BB/J003913/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2012 
End 06/2017
 
Title High resolution melting for mutation discovery 
Description Uses high resolution melting of heteroduplex PCR products to identify natural or induced mutation in wheat. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact We and our collaborators (from UK, Ireland, Italy, Germany, China and others) have used HRM to identify mutations in important wheat genes controlling crop architecture, disease resistance, starch accumulation and quality, fibre levels and other traits. In collaboration with plant breeding companies, these are being introduced into commercial varieties wheat with novel properties. 
 
Description Identification of GA genes in durum wheat 
Organisation University of California, Davis
Country United States 
Sector Academic/University 
PI Contribution Provided bread wheat sequences for screening a durum wheat sequence database.
Collaborator Contribution Identified durum wheat sequences. Carried out qRT-PCR to confirm expression patterns
Impact Sequences for genes of the GA pathway in durum wheat. Published in BMC Plant Biology: Pearce, S., Huttly, A.K., Prosser, I.M., Li, Y.D., Vaughan, S.P., Gallova, B., Patil, A., Coghill, J.A., Dubcovsky, J., Hedden, P., and Phillips, A.L. (2015). Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family. Bmc Plant Biology 15.
Start Year 2012
 
Description TILLING for disease resistance 
Organisation RWTH Aachen University
Country Germany 
Sector Academic/University 
PI Contribution Hosted a visitor who screened the wheat TILLING population for mutations in Mlo genes. Also subsequently provided information derived by TILLING-by-sequencing by custom analysis of exome captures.
Collaborator Contribution Crossed mutations in A B and D copies of MloI to obtain mildew-resistant wheat lines.
Impact Mildew-resistant lines of wheat (non-GM)
Start Year 2012
 
Description TILLING for novel architectural traits 
Organisation University College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution Provided sequence information for wheat BRI1 genes and information on mutations within a Cadenza-EMS population using exome capture
Collaborator Contribution Taking the mutant lines forward for analysis.
Impact Mutant lines of wheat with lesions in brassinosteroid signalling
Start Year 2012
 
Description TILLING in durum wheat 
Organisation John Innes Centre
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provided a protocol for detection of deletions in EMS-mutagenised populations of wheat
Collaborator Contribution Carried out exome capture of an EMS_mutagenised population of bread wheat and analysed for
Impact Deletion data on durum wheat EMS population. Will be published shortly.
Start Year 2012
 
Description TILLING in durum wheat 
Organisation University of California, Davis
Country United States 
Sector Academic/University 
PI Contribution Provided a protocol for detection of deletions in EMS-mutagenised populations of wheat
Collaborator Contribution Carried out exome capture of an EMS_mutagenised population of bread wheat and analysed for
Impact Deletion data on durum wheat EMS population. Will be published shortly.
Start Year 2012
 
Title Development of a web portal for identification of tilling mutants in wheat 
Description Exon capture and sequencing of 1200 EMS-mutagenized lines within the project identified over 5 million mutations in coding regions of genes. These are publicly accessible via a web portal (www.wheat-tilling.com) and users can identify mutants and request seeds. Commercial wheat breeders pay a one-off licensing fee for freedom-to-operate. So far 270 lines have been licensed for commercial development. 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact In addition to commercial users, many hundreds of lines have been supplied to academic wheat biologists world-wide.