TILLING: MODERN TECHNIQUE COMBINES TRADITIONAL MUTAGENESIS AND FUNCTIONAL GENOMICS

Abstract

The information revolution of DNA sequence has created a unique opportunity to investigate the function of genes. The approach that determines the function of genes first defined by DNA sequence analysis is called (Reverse Genetics=RG). The tools available for RG of plants include the use of T-DNA for gene tagging and the use of RNA interference. While these powerful methods, still have limitations, for example, they do not work in all plant species or genera. Targeting Induced Local Lesions IN Genomes (TILLING) is reliable and widely applicable. (TILLING) combines chemical mutagenesis with mutation screens of pooled PCR products, followed by isolation of mutant alleles of the targeted genes. Genes are amplified by PCR using pooled genomic DNA from several individuals as a template. Following denaturation and renaturation of the amplified DNA, heteroduplexes form if organisms with wild type and mutant sequence are both present in the pool. The heteroduplexes can be detected by cleavage with an endonuclease and resolution of the resulting fragments on a sequencing gel. (TILLING) has two significant advantages over existing plant gene knock-out tools: first, it is applicable to any plant since it does not require transgenic or cell culture manipulations. Second, it produces an allelic series of mutations including (Hypomorphic) alleles that are useful for genetic analysis. The (TILLING) technique can be used to discover and survey natural variation. The technique is called (ECOTILLING) and has been applied to human and many plant species. (TILLING) can also be used to detect naturally occurring single nucleotide polymorphisms (SNP’s) in genes among cultivars or ecotypes of any species or genera. These SNP’s can serve as genetic markers in mapping, breeding and genotyping and can provide information concerning gene structure, linkage disequilibrium, population structure or adaptation. The latter, has a prime importance for all crop plants.