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.
