Development of the Lightscanner as a generic platform for novel allele discovery.

Crop improvement through plant breeding relies on having access to suitable genetic variation in the relevant traits. However, domestication has led to a narrowing of the genetic base of many crops. This is certainly true of bread wheat, which arose by rare hybridisation events between wild grasses to yield the modern hexaploid species. While novel genes can be brought in from exotic germplasm sources such as old varieties, landraces and wild species, it is difficult, if not impossible, to assess the potential of such genes in their native background, particularly for complex traits such as yield and grain quality. An alternative strategy for increasing genetic variation involves random mutation of elite germplasm using chemicals or irradiation; many modern varieties of crops, such as 'Golden Promise' barley, have their origins in mutagenesis programmes. There are two strategies for identifying promising mutant alleles: the most direct approach is to select individual plants on the basis of their 'phenotype' / their observable physical characteristics. This 'forward genetic' strategy is also the basis of traditional plant breeding. However, for polyploid species such as wheat, there are multiple copies of each gene; mutations in one copy may have no effect on phenotype and thus individuals cannot be selected on this basis. The TILLING technology, developed at the University of Washington in Seattle, circumvents this problem by identifying promising mutants at the DNA level. For polyploid species, mutants in each copy of the gene can be identified in the mutated population and these different alleles brought together by crossing, to yield a plant with novel properties due to the accumulated mutations. This non-GM technology promises to revolutionise plant breeding, particularly for traits whose genetic basis is well understood, enabling the selection of mutations in specific genes with some confidence of beneficial effects. However, the TILLING method itself is difficult and laborious; in this project we aim to develop a simpler, cheaper method of identifying mutations in specific genes, which also involves alternative technology and so allows freedom to operate outside potential patent constraints. This will be established for wheat, our target crop, but will be applicable to other model and crop species.

The TILLING protocol has been established at RRes to identify novel alleles in candidate genes associated with important traits in wheat. The current protocol uses mismatch cleavage of genomic PCR products from wild-type and mutant alleles followed by denaturing polyacrylamide gel electrophoresis. While this can be very sensitive and capable of high throughput, the method is expensive, laborious and difficult to establish as a robust detection platform. We recently conceived an alternative method of mutation discovery using high-resolution melt curve analysis to identify point mutations in heteroduplex DNA. We propose to establish this new method on the Lightscanner platform in order to assess the feasibility, utility and throughput of the technique for high throughput mutation detection in wheat. We also anticipate that the technique will be transferable to other species and will be particularly useful in diploid organisms where low mutation rates demand high rates of throughput.