Target-selected mutant screen by TILLING in <i>Drosophila</i>

Winkler, Sylke; Schwabedissen, Anja; Backasch, Dana; Bökel, Christian; Seidel, Claudia; Bönisch, Stefanie; Fürthauer, Maximilian; Kuhrs, Antje; Cobreros, Laura; Brand, Michael; González‐Gaitán, Marcos

doi:10.1101/gr.3721805

Cited by 101 publications

(70 citation statements)

References 19 publications

(17 reference statements)

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“…This preponderance of G/C to A/T transitions is consistent with previous studies of EMS-induced mutations in Drosophila (Bentley et al 2000;Winkler et al 2005) and is expected on the basis of the guanine alkylating activity of EMS. Nevertheless, TILLING of EMS-mutagenized Arabidopsis lines resulted in 99.5% G/C to A/T transitions, suggestive of species-specific differences in repair of EMS-induced lesions (Greene et al 2003).…”

Section: Resultssupporting

confidence: 79%

“…In the past, screening was done during the initial generations following mutagenesis, allowing for the recovery of lesions before interbreeding causes loss by selection or drift. In two previous reports, several thousands of lines were screened within a few generations following mutagenesis (Bentley et al 2000;Winkler et al 2005). This strategy was practical for the focus on a small number of targets and to minimize efforts involved in long-term maintenance of thousands of lines.…”

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confidence: 99%

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Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

et al. 2008

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Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, longterm storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for .100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a userfunded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.

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Section: Resultssupporting

confidence: 79%

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confidence: 99%

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

et al. 2008

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“…TIL-LING is a reverse genetic strategy that combines traditional mutagenesis with high-throughput discovery of single-nucleotide changes 2,3 . The method is general and, following its original application to the model plant Arabidopsis thaliana, has been applied to a variety of plant and animal species including maize, lotus, barley, wheat, Drosophila and zebrafish [4][5][6][7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

A protocol for TILLING and Ecotilling in plants and animals

et al. 2006

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We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

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“…The development of TILLING (targeting induced local lesions in genomes) techniques, which can detect singlenucleotide mismatches in heteroduplexes of PCR amplicons, made EMS mutagenesis a workhorse technique for the study of recalcitrant metabolic and signaling processes in plants, animals, and microorganisms (Greene et al, 2003;Winkler et al, 2005;Gilchrist et al, 2006). In sorghum, a few mutated genes have been identified by TILLING based on individual amplicons Blomstedt et al, 2012;Krothapalli et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses

et al. 2016

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Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches.

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Target-selected mutant screen by TILLING in Drosophila

Cited by 101 publications

References 19 publications

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

A protocol for TILLING and Ecotilling in plants and animals

A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses

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