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

Martín B, Ramiro M, Martínez-Zapater JM, Alonso-Blanco C - BMC Plant Biol. (2009)

Bottom Line: 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.Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid-28049, Spain. bmartin@cnb.csic.es

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2803491&req=5

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.

Mentions: The quality of this collection for mutant discovery was evaluated after the analysis of 14 gene fragments distributed among four of the five A. thaliana chromosomes and chosen from requests by different research laboratories (see Methods; Figure 2 and Table 2). These fragments have a GC content similar to that of fragments studied in Col background TILLING collection [10] and to average coding regions of A. thaliana genome [11]. On average, amplicons were 1.1 kb long and 62.2% corresponded to exon sequence, which is similar to genome average exon proportion [11]. In total, we found and confirmed by sequencing 450 mutations in the 15.7 kb analyzed from different amplicons. All mutations corresponded to G/C to A/T transitions, in agreement with the nearly unique type of nucleotide substitutions observed in previous analyses of EMS-mutagenized A. thaliana plants [10]. The distribution of mutations among the EMS doses of the lines was independent of the number of lines per dose (Table 1; χ2 = 28.9; df = 4; p < 0.0001). A larger number of mutations were found at 30, 35 and 40 mM than expected from the number of lines, while the opposite was found at 25 mM. In addition, 34 lines carried several mutations in the same or two different gene fragments (Table 1). An excess of these lines was found in plants derived from high EMS concentrations (≥30 mM) while a deficiency appears in low EMS concentrations (≤25 mM) when comparing with the expected number according to the proportion of lines from each EMS dose (χ2 = 5.8; df = 1; p = 0.016). Therefore, the higher the EMS concentration used to obtain the lines, the larger the number of mutations per line.


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

Martín B, Ramiro M, Martínez-Zapater JM, Alonso-Blanco C - BMC Plant Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803491&req=5

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.
Mentions: The quality of this collection for mutant discovery was evaluated after the analysis of 14 gene fragments distributed among four of the five A. thaliana chromosomes and chosen from requests by different research laboratories (see Methods; Figure 2 and Table 2). These fragments have a GC content similar to that of fragments studied in Col background TILLING collection [10] and to average coding regions of A. thaliana genome [11]. On average, amplicons were 1.1 kb long and 62.2% corresponded to exon sequence, which is similar to genome average exon proportion [11]. In total, we found and confirmed by sequencing 450 mutations in the 15.7 kb analyzed from different amplicons. All mutations corresponded to G/C to A/T transitions, in agreement with the nearly unique type of nucleotide substitutions observed in previous analyses of EMS-mutagenized A. thaliana plants [10]. The distribution of mutations among the EMS doses of the lines was independent of the number of lines per dose (Table 1; χ2 = 28.9; df = 4; p < 0.0001). A larger number of mutations were found at 30, 35 and 40 mM than expected from the number of lines, while the opposite was found at 25 mM. In addition, 34 lines carried several mutations in the same or two different gene fragments (Table 1). An excess of these lines was found in plants derived from high EMS concentrations (≥30 mM) while a deficiency appears in low EMS concentrations (≤25 mM) when comparing with the expected number according to the proportion of lines from each EMS dose (χ2 = 5.8; df = 1; p = 0.016). Therefore, the higher the EMS concentration used to obtain the lines, the larger the number of mutations per line.

Bottom Line: 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.Analysis of TILLer collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of A. thaliana.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid-28049, Spain. bmartin@cnb.csic.es

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.

Show MeSH
Related in: MedlinePlus