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

Abstract

Background: 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. 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.

Results: By 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.

Conclusions: Analysis 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.

Figure 1

Dose effects of EMS mutagenesis in A. thaliana M₁ Ler plants. A) Frequency of M₁ seed germination. B) Frequency of M₁ albino chimeras at vegetative stage. C) Frequency of sterility, embryo lethality and fertility measured as the percentage of different fruit classes. Fruits of M₁ plants are classified as As, Aa, B and C (from fully sterile to nearly normal fertility) depending on the proportion of aborted seeds (see Methods for details). Data are mean ± SE of three to six replicates. Untreated control plants showed 100% germination, 0% albino chimeras and 0, 3 and 97% of A, B and C fruit classes.

Figure 2

Gene fragments analyzed and mutations found in TILLer collection. Positions of silent, missense, splicing site and nonsense mutations are indicated by grey arrow heads, black arrow heads, grey asterisks and black asterisks, respectively.