Development and validation of a flexible genotyping platform for wheat

Developing new strategies to manipulate yield and pest and disease resistance by marker-assisted selection (MAS) underpins the UK's strategy to generate improved wheat varieties. Academic laboratories, genotyping service providers and breeding companies use MAS to track the inheritance of a host of loci controlling desirable traits such as disease resistance, drought tolerance and yield. Until recently most laboratories have used microsatellite markers in their MAS projects, while these markers continue to be used, for many species Single Nucleotide Polymorphisms (SNPs) have become the marker of choice due to their ease of use and scoring and their ability to be automated with relative ease. However, in allohexaploid wheat the task of identifying similar useful sequence polymorphisms is problematical due to the occurrence of homoeologs from the A, B and D genomes. In this proposal, in collaboration, the John Innes Centre, IDna Genetics and KBioscience and Bristol University will develop both a wheat SNP database and a flexible genotyping platform for wheat. The utility of both will be validated by applying the tools and technologies to several exemplar projects. If successful users will be able to access the technology via two routes: The first consists of an in-house set-up requiring only basic molecular biology equipment (PCR machine and fluorescence plate reader). In this case, users would use the database to help them decide which assays to run and which primers/reagents (all publicly available through the database) to order and suggestions as to where these might be obtained. This constitutes a very low barrier for adoption as most users will be able to perform these SNP assays in house at the scale that best fits their purpose. This level of platform will be employed for the work carried out at JIC. The KASPar platform will replace JICs existing SSR genotyping service and, like the existing service, will be available for external work. An aliquot of the full set of KASPar primers will be kept at JIC and made available via this service. The second form of access consists of outsourcing to competitive service providers, such as KBioscience and IDna Genetics. Both companies have high-throughput platforms installed which will help lower costs and both will be set up to run all the KASPar assays developed in this project. The user would only need to provide genomic DNA and a list of which assays to run. In addition to KASPar, members of the community will be able to use the database to access a range of alternative genotyping technologies such as Illumina GoldernGate/Infinium/SureSelect, etc. For instance, the database will enable researcher to either design suitable probes for ordering from the appropriate technology supplier or they will be provided with details as to who offers the required technology on a commercial bases. This database will then be updated until at least 2017.

The 2009 Royal Society report 'Reaping The Benefits' said 'The challenge of a growing population is compounded by new threats, such as pests, diseases and climate change, and this further indicates the need for constant research into new varieties and practices ('running to stand still')'. The report goes on to say 'The biggest gains from technology, come from combinations of improved crops and improved practices'. This proposal, we intend to establish an interactive web-based genotype database that collates the available and accumulating SNP/genotyping information, and provides further related information, for instance map location, primer sequences and genotyping conditions, useful to the wheat community. The information contained within the database will consist of the following: a. Sequence information for each SNP locus across at least 2 different genotypes. b. Primer sequences for use in a variety of assays, for instance Taqman, KASPar, GoldenGate, SureSelect, etc. c. Where appropriate assay conditions and where genotyping services might be available. d. Map location based upon both genetic and deletion map data. e. Genotyping data, including graphical genotypes across at least the 46 varieties, information on haploytpes, rare alleles and population statistics, including data associated with LD-studies as this becomes available. f. Syntenic association data with Brachypodium and rice. In addition we will also, via collaboration with KBioscience and IDna Genetics, validate the SNPs within a commercial environment to assess their Efficacy, Scalability, Flexibility, Versatility and Ease. Finally, we will exploit the KASPar technology in various scientific applications designed to test the procedure against scientific goals of interest to the wheat community.

Wheat is fundamentally important to world agriculture and food security. In 2007 the world harvest of wheat was ~ 550 m tonnes. DEFRA reported that in 2007 ~11% of UK arable land (1,815,900 hectares) was planted with wheat generating ~13,074,480 tonnes with a farm gate value of £2.7 billion. UK breeders and farmers have been very successful in developing/growing wheat varieties with high yield potential, with increases in average UK yields from ~3 tonnes per hectare in 1948 to ~8.0 tonnes in 2006. However, wheat production has not kept pace with demand. Furthermore, wheat productivity is threatened by pests and disease, competition for high quality land, resource limitations, and adverse environmental conditions that dramatically reduce optimal yields. In Europe productivity needs to double to keep pace with demand and to maintain stable prices. Therefore, by narrowing the gap between theoretical maximal yields and actual yields, and increasing potential yields and quality, sustainable/adequate production of one of the world's most important crops could be secured. Who will benefit from this research? 1. Farmers, plant breeders, millers/bakers: Newly developed wheat varieties that fail to either make the recommended list or that fail to gain profitable market share, represent a potential waste of time and resources to the companies involved in their development. In addition, once planted wheat that fails, for whatever reason, to make the bread making quality threshold is usable only as animal feed, incurring a drop in price of up to 25% (£34.5 / tonne, average 2009). Such developments affects the price of feed wheat and increases grain imports as the milling industry must source quality wheat from overseas. Establishment of validated molecular markers for key genomic region conferring, for instance, durable disease resistance or increased grain yield will enable breeders to generate and select improved lines from elite UK germplasm more rapidly than is currently possible. More extensive use of MAS will also enable enrichment for traits which are presently directly selected in breeding programs, such as efficient nitrogen use. This will help maintain farm incomes and improve sustainability of the UK arable and milling industries as well as reducing dependence on imported grain and associated transport costs. Improved yield stability in variable weather conditions in UK wheat will enhance reliability and competitiveness in overseas markets. 2. Scientists and plant breeders: Identification of biological processes linked to agronomic traits and use of genetic/genomic information from model/other cereal systems will help define candidate genes. Detection of genetic variation in existing material, and an enhanced understanding of variation across the genome region, will lead to further improvement in yield and disease resistance. The development and wide use of gene based markers in wheat is a crucial first step in the positional cloning of key genes for UK agriculture which is the route to genomics led predictive wheat breeding. A new era of wheat breeding that, with the funding of this work, the UK would be well placed to lead. How will they benefit from this research? The proposed research will provide both large numbers of easy to use molecular markers and the genotyping data required to apply the markers in a range of circumstances. In addition, by including genotyping companies in our proposal we will also provide a mechanism by which the markers can be used by all members of the community. Such tools and resources will help us to define structure/function relationships for genomic regions and candidate genes, allowing a predictive use of molecular markers for use in targeted breeding programmes to increase yield,disease resistance, abiotic stress tolerance, and grain quality.