Role of reverse genetic approaches TILLING and EcoTILLING in crop improvement: A review

Abstract

Forward and reverse genetics are the two distinct approaches for establishing a connection between an individual's genetic makeup and their observable traits. Targeting Induced Local Lesions in Genome (TILLING) a reverse genetic method, combines chemical mutagenesis with PCR-based screening to pinpoint mutations in regions of interest (McCallum et al., 2000). In TILLING, a specific endonuclease is employed to detect either artificially induced or naturally occurring DNA polymorphisms in a gene of interest. This approach involves introducing mutations into DNA, leading to changes in observable traits due to the resulting point mutations, such as insertions or deletions. The core principles of TILLING include the creation of a population of organisms mutagenized with a chemical called EMS, the use of PCR primers to target the gene of interest, and the identification of rare mutants within this population. A similar technique called EcoTILLING exists, differing only in that it seeks out natural genetic polymorphisms rather than induced ones across various germplasm (Comai et al., 2004). One of the primary advantages of TILLING is its capability for genetic mapping through linkage association analysis and its ability to identify a spectrum of genetic variations, all while remaining cost-effective. ECO-TILLING serves fundamental purposes such as mapping, association analysis, mutation profiling, and the study of biodiversity. TILLING also allows for the efficient, simultaneous screening of multiple genes and enables the prediction of the expected number of genetic variations based on mutation frequency and library size. In summary, TILLING is a highly relevant technique in reverse genetics, effectively complementing the array of available methods. It serves as a valuable, non-GMO approach in molecular breeding, particularly when the desired outcome can be achieved through gene mutations without the complete knockout of the gene.