Tomato TILLING Technology: Development of a Reverse Genetics Tool for the Efficient Isolation of Mutants from Micro-Tom Mutant Libraries

Abstract

To accelerate functional genomic research in tomato, we developed a Micro-Tom TILLING (Targeting Induced Local Lesions In Genomes) platform. DNA pools were constructed from 3,052 ethyl methanesulfonate (EMS) mutant lines treated with 0.5 or 1.0% EMS. The mutation frequency was calculated by screening 10 genes. The 0.5% EMS population had a mild mutation frequency of one mutation per 1,710 kb, whereas the 1.0% EMS population had a frequency of one mutation per 737 kb, a frequency suitable for producing an allelic series of mutations in the target genes. The overall mutation frequency was one mutation per 1,237 kb, which affected an average of three alleles per kilobase screened. To assess whether a Micro-Tom TILLING platform could be used for efficient mutant isolation, six ethylene receptor genes in tomato (SlETR1–SlETR6) were screened. Two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) that resulted in reduced ethylene responses were identified, indicating that our Micro-Tom TILLING platform provides a powerful tool for the rapid detection of mutations in an EMS mutant library. This work provides a practical and publicly accessible tool for the study of fruit biology and for obtaining novel genetic material that can be used to improve important agronomic traits in tomato.

Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase

“…Because point mutations induced by EMS are distributed randomly over the whole genome, allelic series that include both loss-of-function and weak and strong hypomorphic alleles can be found for each target gene (Just et al, 2013). Tomato EMS mutant collections can be further exploited by the targeting-induced local lesions in genomes (TILLING) approach, which allows the discovery of unknown point mutations in known candidate genes (Okabe et al, 2011), or through forward genetic approaches aiming at identifying the mutation underlying the mutant trait. Less straightforward than TILLING, this approach relies first on the phenotypic characterization of the mutant trait and then on the identification of the mutation through map-based cloning, combined (or not) with the candidate gene approach or, more recently, through whole-genome sequencing (Abe et al, 2012;Just et al, 2013).…”

Genetic Diversity in Tomato (Solanum lycopersicum) and Its Wild Relatives

“…A chemical reagent (Ethylmethane Sulphonate) is used to induce genetic mutations. Collections of tomatoes carrying artificially induced genetic variants, called mutant libraries are currently available (Menda, Semel et al 2004;Minoia et al 2010) or under development (Okabe, Asamizu et al 2011). In contrast to transgenic methods, mutagenesis is random, cost effective and is not submitted to GMO regulation.…”

Targeted mutation breeding of horticultural plants

“…Prior to selfing, tomato lines with missense PG mutations were backcrossed twice to reduce background mutations. Delayed ripening in tomato was also obtained by TILLING for induced variation in genes for expansin 1 (Colbert et al, 2011), β-galactosidase 4 (Hurst et al, 2011), and two ethylene receptors (Okabe et al, 2011). Candidate genes for other fruit traits such as color and sugar content are being investigated in EMS-mutagenized populations of tomato Gady et al, 2009;Gady et al, 2011) and melon (Gonzalez et al, 2011).…”