TILLING IN THE SERVICE OF PLANT IMPROVEMENT

Abstract

A major prerequisite for description and functional characterization of the gene pool that contributes to a certain trait or constitutes the genome of a plant species is information about alleles and their combinations (haplotype) among the genes involved. For identification and characterization of novel and a virtually unlimited number of alleles in a relatively high number (hundreds) of genes a high-throughput method is required, which ideally also circumvents sequencing of individual alleles. The recent reverse genetics tool TILLING (‘Targeting Induced Local Lesions IN Genomes’) fulfils these criteria. Originally developed for fast identification of large numbers of alleles in a genotype saturated with induced mutations (McCallum et al. 2000), the latest improvement of TILLING, EcoTILLING (Comai et al. 2004) is especially useful for description of natural allelic variants, which represent a certain group of genotypes or even a whole species. The technique consists of several steps, which will be presented in detail. 1. Selection of primers specific for the genes to be characterized can be performed with a platform of bioinformatic softwares. The analysis includes determination of exon-intron structures, identification of genic regions with high frequency of nonsynonymous mutations, and design of best-fitting primers. 2. Mixing PCR products obtained with each selected genotype to a common standard results in the formation of heteroduplexes that contain mismatches at the position of allelic differences (point mutations, short indels and inversions, etc.). 3. Digestion of heteroduplexes by a mismatch-specific nuclease (e.g. CELI of celery) produces fragments of different length, which are separated by gel electrophoresis and detected by laser excitation of IR labeled primers. Since 50-100 samples are typically analysed on one gel, high numbers of alleles can be identified in a short time. By combination of size standards and image analyis the position of allelic differences can be located at a high accuracy (max. 1-2 nucleotide resolution) without resequencing. An important application of TILLING is the identification of novel alleles in useful genes at a hitherto unknown number and speed, while EcoTILLING may prove useful in dissecting taxonomical and evolutional relationships in higher plants more precisely than ever. Examples will be presented for both applications.