Reverse Genetics with TILLING in Phytophthora sojae

The reverse genetic method TILLING (Targeting Induced Local Lesions in Genomes) is being applied to the plant pathogen Phytophthora sojae. The objective is to recover gene-specific mutants carrying allelic series and/or knockout induced mutations.

A library of 3000 mutant individuals was generated using the chemical mutagens ethylmethanesulfonate (EMS) or ethylnitrosourea (ENU). Gene-specific induced mutations are detected by screening a mirror library of genomic DNA. PCR is used to amplify and fluorescently label 1kb portions of specific genes from the mutant library and the PCR products are then heated and cooled slowly to form hetero- and homoduplexes.

The PCR fragments are treated with the CEL 1 enzyme, a single strand specific endonuclease, and the treated fragments resolved on a LI-COR gel analyzer system. Amplicons harboring induced SNPs are cleaved at the site of the mutation thus producing novel fragments. To date we have identified nine SNP’s in the Necrosis Inducing Protein (NIP) gene in P. sojae using TILLING. Although beyond the scope of this work, the generation of recombinant sexual progeny to recover mutants homozygous for these mutations is underway.