Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis
July 16th, 2019
Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.
This protocol details the development of robust TILLING populations with high mutation frequency by EMS mutagenesis in small grain crops. TILLING populations can be used for functional genomics, as well as for forward genetic space gene discovery in small grain crops. The TILLING populations developed using this technique have high mutation frequencies, and this protocol can be applied to any genotype.
The Cel-1 assay based mutation detection can be conducted using basic lab equipments. The most important step is to determine the optimal EMS concentration. Therefore, an EMS dosage curve should be made specifically for the individual population.
To begin, soak 100 seeds with the genotype of interest in each of six 250 milliliter glass flasks, containing 50 milliliters of distilled water. Shake at 100 RPM for eight hours at room temperature for imbibition. Then, in a fume hood, prepare 50 milliliters of EMS solutions of five different concentrations by mixing EMS liquid and distilled water.
After imbibition, decant the water out of five flasks. Add 50 milliliters of EMS solution in each of the five flasks containing imbibed seeds. Shake the flasks for 16 hours at 75 RPM and room temperature.
Now, decant the EMS solution into an empty waste bottle and pour the treated seeds onto cheesecloth placed on an empty waste bottle to collect separately for each treatment. Use extra water to help pouring of the seeds. Also, spray several milliliters of EMS inactivating solution onto the wall of contaminated flasks.
And immerse used pipette tips in the solution for 24 hours. Inactive the used EMS solution by adding one volume of EMS inactivating solution to treat for 24 hours. Using a twist tie, hold the EMS treated seeds inside the cheesecloth and wash under running tap water for two hours.
After washing, transplant each seed individually into root trainers containing potting soil. Grow plants at 20 to 25 degrees Celsius for a 16 hour light period. After 15 days of transplantation, check the plants and count the number of seeds that failed to germinate.
Record data of plant survival. If the survival rate is not within 40 to 60%conduct a second round of dosage optimization with a modified concentration until achieving the desired lethality rate of 40 to 60%After the mutagenesis experiment, collect the leaf tissue of M2 plants in a 96 well tissue collection box. Tissue is collected from M2 plants which were grown from selfed M1 plants.
One M2 plant is shown from each M1 plant. Extract DNA using a plant DNA extraction kit with a DNA purification system following the manufacturer's recommendations. Then, load two microliters of the DNA extract into LV plates with 16 slots.
Quantify DNA using a spectrophotometer at wavelengths of 260 and 280 nanometers. Dilute the DNA concentration to 25 nanograms per microliter with nuclease-free water. Create four times DNA pools of 200 microliters by combining the DNA from each well in the four 96 well blocks into a pool plate while maintaining the row and column identity of each sample.
Next, prepare a PCR master mix tube for gene specific primers by adding into each tube PCR buffer, forward and reverse primers, and DNA polymerase according to the manuscript. Aliquot the master mix into the wells of a 96 well PCR plate. Then, add the pooled DNA template.
Load the PCR tubes into a thermal cycler, and use a touchdown profile to run the PCR reaction on the thermal cycler. To generate heteroduplexes between mismatched DNAs, incubate PCR products in the thermal cycler in a different program. Then, add 2.5 microliters of homemade Cel-1 endonuclease to each of the heteroduplexed PCR products.
Incubate for 45 minutes at 45 degrees Celsius. After that, terminate the Cel-1 reaction by adding 2.5 microliters of 0.5 molar EDTA at pH eight. Load 30.5 microliters of each Cel-1 treated product mixed with five microliters of dye onto a 3%agarose gel and run at 100 volts for two and a half hours.
To deconvolute mutant pools, determine the zygosity of mutants by running two PCR reactions for individual M2 DNA in which the first reaction contains 2.5 microliters of M2 DNA and 2.5 microliters of wild type DNA and the second reaction contains only five microliters of M2 DNA. This protocol shows EMS mutagenizing of small grain crops and the characterization of mutant. The dosage curve indicates optimal EMS doses for the desired 50%survival rates for three different species of wheat.
The presence of easily identifiable phenotypes in the M2 population confirms effectiveness of mutagenesis in small grain populations. Here are four examples of the mutant phenotypes in M2 TILLING populations, an albino mutant in a barley M2 population, a chlorina mutant in a barley M2 population, a variegated mutant with pink discoloration in an Aegilops tauschii M2 population, and a low-tillering mutant in a Triticum monococcum M2 population. A mutant identification using Cel-1 on agarose gel platforms shows a potential mutant pool identified out of 12 mutant pools by unique cleaved bands.
The deconvolution of mutant pools determined the zygosity of mutation and helped to track the mutation down to individual samples. The A4 pool had heterozygous mutations, indicated by cleaved bands in both Box 4 and Box 4 wild type DNA samples. The H5 pool contained homozygous mutations as unique cleaved bands were only present in Box 5 H5 wild type DNA sample.
The most important step is to determine the optimal concentration of EMS to achieve 40 to 60%lethality rate. Once a TILLING population with a high mutation frequency has been developed it can be used for functional characterization of any gene of interest. Additionally, the population can be a source of useful genetic variation for crop improvement.
TILLING has been used extensively for functional genomic studies in model and crop plants. The range of mutations obtained can be missents, knockouts, silent, or even misspliced forms. EMS is a mutagen.
Therefore, use appropriate personal protective equipment while handling the EMS. Decontaminate leftover solutions and used pipette tips.
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Overview
This protocol details the development of robust TILLING populations with high mutation frequency by EMS mutagenesis in small grain crops. TILLING populations can be used for functional genomics and forward genetic space gene discovery.
Key Study Components
Area of Science
- Genetics
- Plant Biology
- Mutagenesis
Background
- TILLING is a reverse genetics tool used to identify mutations.
- EMS is a chemical mutagen that induces point mutations.
- Small grain crops are important for food security.
- High mutation frequency is essential for effective gene discovery.
Purpose of Study
- To develop TILLING populations in small grain crops.
- To enhance functional genomics research.
- To facilitate forward genetic studies.
Methods Used
- Ethyl methanesulfonate (EMS) mutagenesis.
- Development of EMS dosage curves for optimal concentration.
- Cel-1 assay for mutation detection.
- Application to various genotypes.
Main Results
- High mutation frequencies achieved in TILLING populations.
- Protocol applicable to any genotype.
- Basic lab equipment sufficient for mutation detection.
- Optimal EMS concentration is crucial for success.
Conclusions
- The protocol provides a reliable method for TILLING in small grains.
- It supports functional genomics and gene discovery efforts.
- Future applications can enhance crop improvement strategies.
Frequently Asked Questions
Executive Industry Relevance
Developing high-frequency TILLING populations in small grain crops enables functional gene validation where transformation-based methods are limited. This approach supports target validation and phenotypic screening by generating allelic series for mechanistic de-risking. The Cel-1 assay provides a low-resource, reproducible method for mutation detection, facilitating early-stage screening and lead identification in crop improvement pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses through allelic series generation in disease-relevant systems.
- Operational Value: Supports biological de-risking by linking genotype to phenotype in small grain models.
- Scientific Value: Enhances target confidence by enabling functional validation of genes of interest via mutation detection.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream screening using PCR-based Cel-1 assay on agarose gel.
- Operational Value: Ensures assay standardization and reproducibility through optimized EMS dosage curves and 40–60% lethality targeting.
- Scientific Value: Enables quantitative dependent variable measurements via mutant pool deconvolution and zygosity determination.
Translational & Preclinical Research
- Scientific Value: Provides disease-relevant systems for studying gene function in small grain crops with observable mutant phenotypes.
- Operational Value: Supports translational biomarker alignment by connecting molecular lesions to phenotypic outcomes.
- Scientific Value: Facilitates preclinical model development through stable M2 populations derived from selfed M1 lines.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by enabling hypothesis testing, pathway clarification, and biological de-risking prior to lead identification.
- Discovery Biology: Supports hypothesis testing and pathway clarification via targeted mutation induction and detection in target genes.
- Screening: Delivers assay readiness and quantitative outputs through Cel-1-based mutation detection in pooled DNA samples.
- Analytics: Enables comparison of conditions via heteroduplex formation and cleaved band analysis on agarose gels.
- Translational Research: Connects to preclinical continuity through stable mutant lines with identifiable phenotypes for trait validation.
- Enterprise Reuse: Establishes a reusable mutagenesis platform applicable to any genotype for sustained genetic variation generation.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence through high-frequency mutation spectra including missense, knockout, and splice variants.
- Operational Value: Standardization and scalability via dosage curve optimization and basic equipment-dependent Cel-1 assay.
- Strategic Value: Improved go/no-go decisions by reducing mechanistic ambiguity in gene function studies.
- Portfolio Impact: Risk-adjusted prioritization through access to allelic variants for crop improvement and trait validation.
Implementation Considerations
- Requires expertise in mutagenesis, DNA extraction, and PCR-based mutation detection.
- Dependent on thermal cyclers, spectrophotometers, and agarose gel electrophoresis systems.
- Necessitates cross-team standardization of EMS handling, DNA pooling, and Cel-1 reaction protocols.
- Requires adaptation across model systems via genotype-specific dosage curve development.
- Practical limitation: mutation detection sensitivity depends on heteroduplex formation and Cel-1 activity optimization.
