Isolation of pooled selected gene segments for allele discovery by next generation sequencing

Future crop varieties will need improved performance traits for the consumer and the producer, and reduced environmental impact (e.g. lower fertiliser or pesticide input). These traits are currently monitored by 'molecular markers', which are genetic fingerprinting tests. An alternative approach is to sequence the genetic information directly. A revolution in 'next generation sequencing' is currently underway' with speed doubling annually and prices tumbling, Direct sequencing can give far greater accuracy than molecular markers but there is a technical bottleneck caused by the fact that it sequences many individuals in the same container, raising the difficult issue of how to keep track of which sequence belongs to which individual. In this project we propose to develop, test and compare two promising tracking methods, using barley as a test-bed.

Studies of genetic diversity and allele discovery in both plants and animals are currently dominated by molecular markers. High throughput SNP genotyping offers great accuracy and detail that has been successful for population and association genetic studies in humans where up to a million SNPs are available. For most animals and plants this luxury in genomic resource is a very long way away and SNP sets are less effective in describing the higher diversity found in wild or semi-domesticated populations. An alternative approach is to replace marker analysis by next generation sequencing, thus effectively 'genotyping by sequencing'. Direct sequencing can give far greater resolution of haplotypes in diverse germplasm at far lower cost per polymorphism and its efficiency is increasing dramatically. The bottleneck to implementing this is the fact that next generation sequencing samples huge numbers of pooled DNAs in parallel and if you want to sequence lots of samples at lots of loci, then sequence tags are needed to define the genome of origin. This technology is nearly available for capture arrays (e.g. Nimblegen) but needs to established, particularly with regard to tagging the genomes. In addition we have developed a PCR-based method that can in principle achieve a similar result using a few hundred PCRs and primers, potentially saving considerable time and effort. We propose to develop, test and compare both capture arrays and our new method, using barley as a test-bed and to derive optimised protocol(s) for isolating diverse [gene loci x sample] sets for downstream next generation sequencing.