A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis

Beatriz Martín,Carlos Alonso-Blanco,José M Martínez-Zapater,Mercedes Ramiro

doi:10.1186/1471-2229-9-147

Abstract

BackgroundArabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in A. thaliana by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.ResultsBy mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M2/M3 independent mutant lines in the reference strain Landsberg erecta (Ler) of A. thaliana. This population has been named as the Arabidopsis TILLer collection. The frequency of mutations per line was maximized by using M1 plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, Ler population providing a total of more than five million induced mutations. It is estimated that TILLer collection shows a two to three fold higher EMS mutation density per individual than previously reported A. thaliana population.ConclusionsAnalysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana. Comparisons with TILLING populations in other organisms indicate that this new A. thaliana collection carries the highest chemically induced mutation density per individual known in diploid species.

Highlights

Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes
In this work we have developed a new collection of A. thaliana ethyl methanesulphonate (EMS) induced mutants for TILLING reverse genetics, aiming at two major objectives
Generation of a permanent collection of highly EMSmutagenized lines in Arabidopsis To obtain a new population of chemically induced mutant lines useful for reverse genetic studies in Arabidopsis thaliana, seeds of the Landsberg erecta (Ler) glabrous1-1 genotype were mutagenized with EMS at concentrations of 20 to 50 mM (Figure 1)

Summary

Introduction

Arabidopsis thaliana is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. A major challenge in plant biology is the identification of biological functions for all genes from the main model plant species, Arabidopsis thaliana and rice To this end, a large number of genetics and genomics resources are being developed in both model plants [1,2]. An allelic series of induced mutations with different effects on gene function can be isolated by screening a few thousands mutagenized plants [6]

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