Reverse genetics by TILLING

(3) Seeds are mutated to produce M, generation. Each M, plant is heterozygous for mutations in different genes (colors). NI1 individuals Each M individual is propagated to produce an M2 family. I l l l \ Malamllies :1" ”:2 +/+:3:+/.:4 :5: Each M family is segregating for mutations in different genes( omozygous mutants in color). Seed stock and DNA samples are collected from each M family Seed stocks represent a repository of mutants (b) Mutations' In specific genes are identified by analyzing DNA isolated from each M family. For example, one representative M family has a red mutant segregating. DNA is collected and screened for mutations in target gene by PCR amplification. -> I = Target gene PCR products: l Wild-type G Mutant A — — allele allele 1- GHomoduplex DNA G Heteroduplex DNA F _ T__ Endonuclease (Cell) cleaves a single strand at mistakes in geteroduplex DNA. C l Denaturing electrophoresis I 2 3 4 5 Fm Uncut DNA “DC“. ‘mxgy‘mf ! (wild-type) DNA Cut DNA Cut DNA gene of interest.