Targeted mutagenesis in sorghum using an improved high-throughput screening platform – a reverse genetics strategy to complement the sorghum genomics effort
As a natural complement to the Sorghum Genomics effort, our group has undertaken a reverse genetics strategy involving point-mutagenesis, followed by CEL I mismatch screening. EMS-mutated populations have been generated in four white Sorghum lines. Our research has further developed the mutation screening technique of ‘CEL I mismatch detection’. Our modified technique is a significant improvement on the standard protocols (originally optimized for slab gel systems). Our protocol benefits from the increased sensitivity offered by a capillary electrophoresis platform, enabling a larger degree of sample pooling. Throughput has also been improved via a significant reduction in the amount of background detected. In addition, our protocol uses a reduced number of steps, once again improving the efficiency of the mutation screening process. Dosimetry trials were performed and eight treatments were chosen for application across all four lines. Treatments were chosen to represent a range of interactions between concentration (of EMS) and time (of imbibition). The resulting M2 populations will be initially mined for mutations in the genes responsible for starch synthesis and grain quality. Previous work with Maize, a model organism for Sorghum, has suggested that the EMS induced mutation density, will be approximately 2 transitions per megabase. Based on this, a population size of 5000 individuals will be sufficient to deliver a large allelic series in any gene of interest (assuming extant sequence information is available). M2 plants will be grown to maturity and M3 seed collected for phenotypic analysis of the lines of interest. In addition to our starch analysis, the populations may be made available as a resource for the Sorghum research community.
Cross, MJ, Lee, LS, Rice, NF & Henry, RJ 2006, 'Targeted mutagenesis in sorghum using an improved high-throughput screening platform – a reverse genetics strategy to complement the sorghum genomics effort', paper presented to the Plant and Animal Genomes Conference XIV, San Diego, California, USA, 14-18 January.