TILLING & EcoTILLING in tomato : harvesting artificial and natural genetic diversity

Mutations in genes controlling specific traits of interest are widely used in plant breeding to introduce particular characteristics in commercial varieties. The establishment of a large mutant collections and the identification of mutations in candidate genes controling traits of interest represents an important resource for plant breeding. Chemical mutagenesis with EMS produces G/C to A/T transitions and is a well-characterized method to generate a saturated mutant population. The TILLING (Targeting Induced Local Lesions IN Genomes) process enables the identification of mutations in specific genes of interest and produces heteromorphic as well as knock-out alleles. In order to apply the TILLING strategy to tomato, we have created two new populations of 8025 M2 and 6600 M3 lines respectively derived from seed treatment with 1% EMS of Solanum esculentum (var. TPAADASU). To screen the collection for mutations in selected genes, two novel high-throughput SNP detection technologies were adapted. Firstly, Conformation Sensitive Capillary Electrophoresis (CSCE) and secondly, high resolution DNA-melting analysis (HRM). Our results indicate a mutation frequency of 1,1 mutation per 1kb per 1000 plants. Mutations in genes involved in carotenoid biosynthesis and tolerance to salinity stress, Psy1 and ProDh, were identified. Characterisation of the mutant lines, carrying knock-out or amino acid substitution alleles, using plant growth assays, metabolic profiling and gene expression studies allowed the demonstration of the TILLING strategy as an efficient method for plant breeding purposes and plant biology research. EcoTILLING was set up using the HRM based SNP screening method to screen the EU-SOL and CBSG tomato core collections for candidates genes involved in sugar metabolism. 13 haplotypes over 6 target genes were identified and related to the studied trait, total soluble sugars. We show that HRM is a fast and cost effective method to unravel natural genetic diversity in candidate genes.