Is it the end of TILLING era in plant science?

Abstract

Since its introduction in 2000, the TILLING strategy has been widely used in plant research to create novel genetic diversity. TILLING is based on chemical or physical mutagenesis followed by the rapid identification of mutations within genes of interest. TILLING mutants may be used for functional analysis of genes and being nontransgenic, they may be directly used in pre-breeding programs. Nevertheless, classical mutagenesis is a random process, giving rise to mutations all over the genome. Therefore TILLING mutants carry background mutations, some of which may affect the phenotype and should be eliminated, which is often time-consuming. Recently, new strategies of targeted genome editing, including CRISPR/Cas9-based methods, have been developed and optimized for many plant species. These methods precisely target only genes of interest and produce very few off-targets. Thus, the question arises: is it the end of TILLING era in plant studies? In this review, we recap the basics of the TILLING strategy, summarize the current status of plant TILLING research and present recent TILLING achievements. Based on these reports, we conclude that TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects.

Keywords: NGTs; TILLING; crops; functional genomics; mutagenesis; mutations; plants; reverse genetics.

Figure 1

The general flow of traditional TILLING strategy. The first step is the mutagenesis of seeds. The seeds are propagated into further generations and DNA is isolated from M₂ plants individually. The next step is DNA pooling, followed by the PCR reactions for specific amplicons. There are many methods of mutation identification - either direct or based on heteroduplex analysis. After the identification of a particular plant carrying a mutation in a gene of interest, the phenotypic evaluation should be performed to assign the gene function. DHPLC, Denaturing High-Performance Liquid Chromatography; HRM, High-Resolution Melting; CEL I- endonuclease extracted from a Celery Juice Extract; NGS, Next Generation Sequencing.