Developing RAD markers as a resource for plant breeding

In order to improve the ability of plant breeding programmes to deliver the agricultural increases mandated by a growing population and changing climate, new techniques must be developed for rapid discovery and genotyping of genetic markers. The RAD (Restriction-site Associated DNA) sequencing (RADSeq) technique developed by Professor Eric Johnson of the University of Oregon, generates tens of thousands of genetic 'tags' from genomic DNA. Due to the capabilities of modern DNA sequencers, it is possible to sequence each of these tags many times and thus reliably spot genetic differences between two individuals. The capacity of second-generation sequencers is such that tags from multiple individuals can be pooled within a single sequencing run whilst still maintaining a high enough coverage of each tag to identify genetic differences. Tags from each individual can be identified by adding a unique 'molecular identifier' to the DNA prior to sequencing. By carefully selecting the right number of tags to be generated, it becomes possible to screen enough individuals within a single run to cover an entire genetic mapping population. RAD sequencing therefore combines the discovery, genotyping and mapping of genetic markers into a single step. Furthermore, if the phenotype of the samples is known, the data can be used to identify markers which segregate along with the phenotype, assisting in gene mapping and potentially gene identification. To date, RADSeq has primarily been used in animal or microbial systems. We propose to apply the RADSeq technique to a model cereal species, Lolium perenne (perennial ryegrass), in order to determine the applicability of this technique to improving plant breeding efforts. As a test case, we will use an existing mapping population designed to identify the two genetic loci controlling a self-incompatibility system in Lolium (ryegrass). We will perform RADSeq in the parents of this population at high coverage using two tag densities. We will then screen pooled mapping population progeny from each of four segregating genotypes (two per locus) in order to identify RADSeq markers which appear unique to each genotype. Finally, we will use the RADSeq marker information to construct a genetic map for this population and confirm the bioinformatic identification of a small subset of genetic markers using conventional genotyping. The proposed work will enable us to determine how well the RADSeq technique performs as a method for rapid marker discovery and genotyping in crops, using one of the most difficult examples - a highly heterozygous, outbreeding species. If RADSeq performs well under these conditions, it should easily be applicable to other crop systems. The usefulness of RADSeq as a tool for mapping of genetic loci will also be assessed by attempting to map polymorphisms associated with the self-incompatiblity loci of grasses. Identifying these genes is of high importance to grass breeders as they would allow greater control of mating during breeding programmes.

RAD sequencing (RADSeq) is a high-throughput technique for marker discovery/genotyping. The technique generates short sequence reads adjacent to restriction enzyme sites, which can then serve as 'tags' to identify restriction polymorphisms between samples (similar to AFLP) or be mined to identify the presence of SNPs. By choosing a restriction enzyme which cuts infrequently within the study genome, the complexity of even large genomes can be reduced to a level where high-throughput marker discovery/genotyping can be performed using next-generation sequencing. A restriction enzyme is selected to produce 50-250,000 fragments per digest. Given that one lane of a Solexa flow-cell produces ~12 million reads, individual tags will be sequenced multiple times, making it possible to score presence/absence in the same manner as standard AFLP. However, RADSeq possesses the additional benefit of sampling sequence diversity around restriction sites, allowing for SNP discovery. Multiple individuals can be pooled within a single lane (using a unique 5bp molecular identifier for each sample) and the required coverage of tags needed for SNP genotyping will still be achieved. It is here that the power of next-generation sequencing becomes apparent: a whole flow-cell could potentially be used to assay an entire mapping population. We therefore aim to assess the feasibility of adapting the RADSeq technique to marker discovery/genotyping and mapping in crops, using the forage grass Lolium perenne as a model.We will initially test a series of restriction enzymes on the parental genotypes to produce two tag densities and identify parent-specific RADSeq markers, a subset of which will be validated using another genotyping technique. We will then conduct genotyping of a mapping population of 60 segregants and use this data to 1) construct a genetic map of RADSeq markers and 2) identify marker/trait associations for two genetic loci involved in self-incompatibility.

The successful implementation of RAD sequencing (RADSeq) in crops will result in a number of benefits to non-academic users, primarily among the plant breeding community. Development of high-throughput techniques for discovery and genotyping of genetic markers is crucial to improving breeding programmes and RADSeq has considerable potential in this area. RADSeq is also valuable as a technique for rapid construction of genetic linkage maps, given that an entire F2 population can theoretically be screened simultaneously. Gene discovery is therefore enhanced, further benefitting the plant breeding community. The technique also has considerable potential for rapid screening of diverse germplasm collections for exploitation of natural genetic resources in plant breeding. Economically speaking, ryegrass improvement alone would provide benefits - IBERS Lolium varieties alone comprise 28% of the UK market and have an annual retail value of £5.5 million. Contributions to the improvement of agricultural development also impact the general public in the longer term, by increasing the security of the global food supply in the face of a growing population and changing climate. On a more limited scale, identification of the genes involved in gametophytic self-incompatibility in grass species is of high importance to the grass breeding community. Understanding the genetic basis of this mating system will allow for greater control of male transmission during breeding programmes. The outcomes of this research will be communicated to the wider plant breeding community via associative links such as the Monogram Network and QUOATS LINK. Other direct links to grass breeders include a LINK programme including the British Grassland Society (BGS) along with several levy boards (EBLEX, Dairy Co, Hybu Cig Cymru and others). IBERS research is regularly communicated in the BGS newsletters, journals and website. The Grassland Development Centre based in Aberystwyth as part of IBERS also regularly engages in communicating the latest scientific developments to the breeding and farming communities. Finally, IBERS staff attend annual trade fairs such as the European Dairy Event and the National Beef Event where stands are available for poster presentations and direct engagement with members of the farming community. Genetic markers of use for Lolium breeding which are identified via this project will be relayed to breeders via commercial partnerships (such as IBERS' existing partnership with Germinal Holdings, which has resulted in the production of several commercial Lolium varieties, including the high-sugar varieties AberMagic and AberDart). Germinal have European partnerships with other breeding/seed companies such as Jouffray (France) and Steinach (Germany). IBERS also have industrial links to Eurograss, a consortium of German, Danish and Dutch breeding companies, as well as an ongoing reciprocal technology/resources exchange programme with ViaLactia Biosciences Ltd in New Zealand. Syngenta are also involved with IBERS Lolium research, having jointly funded the current Lolium physical mapping project. In addition to these direct interactions with the breeding community, IBERS makes every effort to communicate science to the wider audience via its departmental website and engagement with the local and national press. The institution regularly produces a newsletter, IBERS Innovations, to present our research in layperson's terms and also runs a series of public seminars at our Science Cafe meetings. Agricultural research is traditionally presented at the Royal Welsh Show, where it can be communicated with both farmers and members of the general public.