TILLING - a shortcut in functional genomics

Abstract

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.

Mutagenesis for Crop Breeding and Functional Genomics

“…While endogenous transposons have been used for gene disruptions in some crops such as maize and rice, a major development came in the late 1990s with a reverse genetic approach known as TILLING that uses induced mutations (Meeley and Briggs 1995;McCallum et al 2000;Hirochika 2001;Conrad et al 2008;Hunter et al 2014). TILLING, short for Targeting Induced Local Lesions IN Genomes, typically utilises mutagens that induce a high density of induced mutations randomly throughout the genome (Kurowska et al 2011;Greene et al 2003;Jankowicz-Cieslak et al 2011). A population of between 3000 and 6000 mutant lines can be developed that contains multiple mutations in every gene in the genome.…”

Recent Biotechnological Advances in the Improvement of Cassava

“…TILLING involves induction of mutations in the plant genome using classical mutagenesis approaches followed by traditional or high throughput deep sequencing to identify the mutations in the gene of interest [77][78][79]. This technique has been used in allele discovery in different plant species [80][81][82][83].…”

Self-Incompatibility in the Brassicaceae

“…TILLING is a reverse-genetic method that takes advantages of classical mutagenesis, sequence databases, and high-throughput PCR-based screening for point mutations in a targeted sequence (Henikoff et al 2004 ). The key advantage of TILLING over competing methods is that it can be applied to any plant species, regardless of ploidy level, genome size, or genetic background (Kurowska et al 2011 ). TILLING extends genomic resources, particularly in organisms lacking reverse-genetic tools, where mutants with a range of phenotypic severity are highly desirable.…”