Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

1. Preparation of dosage curve for effective mutagenesis

1. Soak 100 seeds with the genotype of interest in six 250 mL glass flasks (100 in each flask) containing 50 mL of distilled water. Shake at 100 rpm for 8 h at room temperature (RT) for imbibition by the seeds.
2. In a fume hood, prepare 50 mL of 0.4%, 0.6%, 0.8%, 1.0%, and 1.2% (w/v) ethyl methanesulfonate (EMS) solution by dissolving 0.167, 0.249, 0.331, 0.415, and 0.498 mL of EMS in distilled water, respectively.
   NOTE: EMS is liquid at RT with a density of 1.206 g/mL.
   CAUTION: Use appropriate personal protective equipment (PPE) while handling EMS.
3. Decant the water out of five flasks and add 50 mL of EMS solution in each flask containing imbibed seed so that there are six different treatments with 0.0%, 0.4%, 0.6%, 0.8%, 1.0%, and 1.2% EMS solution. Shake flasks for 16 h at 75 rpm and RT.
4. Decant the EMS solution and collect the treated seeds separately for each treatment using cheese cloth. Inactivate the used EMS solution by adding one volume of EMS-inactivating solution (0.1 M NaOH, 20% w/v Na₂S₂O₃) for 24 h. Also treat the contaminated flasks and pipette tips with the EMS-inactivating solution for 24 h.
5. Wash the EMS-treated seeds under running tap water for 2 h. Transplant each seed individually into root trainers containing potting soil.
6. Grow plants at 20–25 °C under a 16 h light period.
7. Record data on plant survival after 15 days of transplantation. Use the following equation to calculate the survival rate for each treatment:
   
   NOTE: If the germination rate is lower than 100% in controls, accurate survival rates in all the treatments should be calculated after subtracting the number of seeds that failed to germinate in the controls. A survival rate of 40%–60% is desirable for effective mutagenesis. It may be required to perform a second round of dosage optimization with a modified concentration according to the survival of the treated seeds until achieving the desirable lethality rate of 40%–60%.

2. Mutagenesis and maintenance of mutant population

1. Soak the final batch of at least 3,000 seeds dividing equally (600 seeds each) in five 1,000 mL flasks containing 300 mL distilled water. Shake for 8 h at 100 rpm under RT for imbibition.
   NOTE: The final size of the desirable population will depend on the mutation frequency and ploidy level of the genotype, but it is advisable to use at least 3,000 seeds for hexaploids, 4,000 seeds for tetraploids, and more than 7,000 seeds for diploids.
2. In a fume hood, prepare 1,500 mL of the optimized EMS concentration solution in distilled water.
3. Decant the water out of the flasks and add 300 ml of the optimal concentration of EMS solution in each flask containing 600 imbibed seeds. Shake the flasks for 16 h at 75 rpm and RT.
4. Decant the EMS and collect the treated seeds in cheese cloth. Inactivate the EMS solution and treatment containers with EMS-inactivating solution as done in step 1.4.
5. Wash the EMS-treated seeds under running tap water for 2 h. Transplant each EMS-treated M₁ seed individually into root trainers.
6. Grow M₁ plants (derived from M₀ seeds) at 20–25 °C under a 16 h light period.
   NOTE: It may be required to vernalize the seedlings at the two leaf stage for 6 weeks at 4 °C, if the genotype of interest has a winter-type growth habit.
7. Allow the M₁ plants to self-pollinate, and harvest the M₂ seeds separately for each fertile M₁ plant.
   NOTE: To avoid chances of potential outcrossing, cover the spikes of M₁ plants with pollination bags before anthesis.
8. Plant a single M₂ seed from each M₁ plant to avoid genetic redundancy.
9. Collect tissue from M₂ plants at the two-leaf stage in 1.1 mL of racked 96 well microtubes. Collect around 80 mm of leaf tissue from each plant and record the ID of each sample in a tissue collection plan.
10. Freeze-dry the leaf tissue using a lyophilizer and store at -80 °C.
11. Maintain the M₂ plants at 20–25 °C under a 16 h light period.
12. Record data on mutant phenotypes of the M₂ plants at regular intervals. The expected phenotypes are albino, chlorina, grassy shoot, variegated, partially fertile, sterile, etc.
13. Allow the M₂ plants to self-fertilize and mature. Separately harvest and save the M3 seeds of the M₂ plants (Figure 1).
    NOTE: M seeds are used for validating the phenotype in reverse genetics studies. Therefore, M should be carefully catalogued and stored under cool and dry conditions. Alternatively, if the seed must be increased in field, head rows can be planted for each M plant, and M seeds from each row can be harvested and saved separately as the TILLING population resource.

3. Cel-1 assay for genetic characterization of mutants

1. Extract DNA from the leaf tissue of M₂ using a plant DNA extraction kit with a DNA purification system (see Table of Materials) following the manufacturer’s recommendations.
2. Quantify DNA using a spectrophotometer and normalize the DNA concentrations to 25 ng/µL with nuclease-free water in 96 well blocks.
   NOTE: It is important to check the quality of the DNA by running it on a gel, as low-quality DNA (smeared DNA) may result in false negatives in pooled samples.
3. Create 4x DNA pools by combining DNA from four 96 well blocks into one plate, while maintaining the row and column identity of each sample. Add 50 µL of DNA from each individual sample into the pool plate so that each pool plate well contains a total 200 µL of DNA from four different 96 well blocks.
4. Catalogue the identity of pooled DNA in the format of Pool Plate-Row-Column (e.g., Pool 1 A1 = Box1A1 + Box2A1 + Box3A1 + Box4A1).
5. Design genome-specific primers [A1] for the gene of interest using the Genome Specific Primers (GSP) page <https://probes.pw.usda.gov/GSP> with default settings for polyploid species. For diploids, use Primer 3 <http://primer3.ut.ee/> for designing primers using default settings. Design multiple primers, if needed, to cover the entire coding region of the gene of interest.
   NOTE: The latest IWGSC assembly can be used to obtain sequences for the gene of interest in wheat using the URGI BLAST tool <https://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST>. The optimal amplicon length is in the range of 800–1,500 bp. Table 1 shows examples of primers for waxy genes in hexaploid wheat.
6. Run a PCR for gene-specific primers on pooled DNA as follows:
   1. Add 5 µL of PCR buffer, 2 µL (each) of 4 µM forward and reverse primers, 0.1 µL of DNA polymerase (see Table of Materials), 5 µL of pooled DNA template, then increase the volume to 25 µL using nuclease-free water.
   2. Use a touch down profile to run the PCR reaction on a thermal cycler as follows: 95 °C for 1 min, seven cycles of 95 °C for 1 min, 67 °C to 60 °C for 1 min with temperature decreases of 1 °C per cycle and 72 °C for 2 min, followed by 30 cycles of 95 °C for 1 min, 60 °C for 1 min, 72 °C for 2 min, and final extension at 72 °C for 7 min.
      NOTE: Above PCR profile works on wheat DNA template for most of the primers designed using primer 3 default settings. In case of non-specific amplification, PCR profile should be made stringent before moving to the next steps.
7. Generate heteroduplexes between mismatched DNA by incubating PCR products in thermal cycler using profile as follows: 95 °C for 2 min, five cycles of 95 °C for 1 s, 95 °C to 85 °C for 1 min with temperature decreases of 2 °C per cycle, and 60 cycles of 85 °C to 25 °C with decreases of 1 °C per cycle.
8. Add 2.5 µL of homemade Cel-1 endonuclease to the heteroduplexed PCR products and incubate for 45 min at 45 °C. Terminate the Cel-1 reaction by adding 2.5 µL of 0.5 M EDTA (pH 8.0).
   NOTE: Cel-1 endonuclease can be extracted from fresh celery stalks using the protocol performed by Till et al.¹⁹ It is very important to test the activity and optimum amount of Cel-1 endonuclease, which can be tested using previously characterized mutants or a commercially available mutation detection kit.
9. Run the Cel-1 treated products on a 3.0% agarose gel at 100 V for 2.5 h. The wells containing smaller and unique cleaved band(s), in addition to full-length uncleaved bands, will contain the mutant DNA sample.
10. Denconvolute mutant pools.
    1. Follow steps 3.6, 3.7, 3.8, and 3.9 for individual M₂ DNA samples constituting the mutant pools identified in step 3.9.
    2. To determine the zygosity of mutants, run two PCR (as described in step 3.6) for individual M₂ DNA, in which the first reaction contains 2.5 µL of M₂ DNA and 2.5 µL of wild-type DNA and the second reaction contains only 5 µL of M₂ DNA. If the mutation is heterozygous, an additional cleaved band will be present in both reactions. On the other hand, additional cleaved bands will be found only in the first reaction if the mutant is homozygous.
11. To identify the nature of the mutation, sequence the PCR products of the confirmed mutants using a Sanger sequencing platform following the manufacturer’s instructions.

4. Calculation of mutation frequency

NOTE: Mutation frequency of a TILLING population refers to the average physical distance in which one mutation occurs in the individuals of that population. For example, a mutation frequency of 1/35 kb in a TILLING population means that an average individual of that population possesses 1 mutation per every 35 kb in the genome.

1. To determine the mutation frequency of a TILLING population, calculate the total number of bases screened.
2. To calculate the total number of bases screened, multiply the PCR product size by the total number of individuals screened.
3. Divide the total number of bases screened by the number of unique mutations observed using the following equation, which will yield the physical region possessing 1 mutation in the given TILLING population:
   
   NOTE: To account for the limitation in resolution of 50 bp at both the ends based on an agarose gel-based platform, subtract 100 bp from the product size in the calculation.