Mutation Induction and Reverse Genetics for Vegetatively Propagated Plants

The development of high-throughput and low-cost methods for the discovery of natural and induced mutations has enabled novel approaches for reverse-genetics and germplasm characterization. Methods that combine enzymatic mismatch cleavage and fluorescence detection by gel or capillary electrophoresis have been the primary mutation discovery platform for the reverse-genetics strategy known as TILLING (Targeting Induced Local Lesions IN Genomes). This approach has worked well in combination with mutagens that cause primarily SNPs or small indels. We describe the adaption of traditional TILLING methods for the induction, recovery and monitoring of inheritance of induced SNP mutations in vegetatively propagated triploid banana. This work provides a foundation for other projects on understudied obligate and facultative vegetatively propagated crops important in agriculture. We are also interested in evaluating the effect of different mutagens on plant genomes that might prove more efficient for developing novel traits for breeding purposes.  For example, approximately half of mutant plant varieties registered in the IAEA’s mutant varieties database (http://mvgs.iaea.org/Search.aspx) are derived from treatment of plant materials with gamma rays. Data suggests that the density of induced SNP and small indel mutations is low, making traditional enzymatic based mutation discovery systems inefficient. We are exploring alternative methods for the simultaneous discovery of a broad spectrum of induced mutations, and evaluating the efficacy of this approach for reverse genetics in vegetatively propagated species such as cassava.