Targeted Mutagenesis for Functional Analysis of Gene Duplication in Legumes

Assessment of gene function oftentimes requires mutant populations that can be screened by forward or reverse genetic analysis. The situation becomes more complicated in polyploidy or paleopolyploid genomes that have two or more copies for most genes. Here we describe a method for engineering zinc-finger nucleases (ZFNs) for the purpose of creating targeted mutations in the paleopolyploid soybean genome. ZFNs are recombinant proteins composed of an engineered zinc-finger array fused to a nonspecific cleavage domain. When engineered to recognize a specific nucleotide sequence, the cleavage domain will generate highly mutagenic DNA double-strand breaks frequently resulting in insertions and deletions at the target locus. Depending on the number of target sites present within the genome, this method has the capacity to target either single- or multi-copy gene families. In this chapter, we describe an inexpensive, rapid, and user-friendly approach for ZFN assembly and application in soybean based on the previously described context-dependent assembly method.