Process Explained

The technology, known as TILLING (Targeting Induced Local Lesions IN Genomes) is a ‘reverse genetics’ process as developed by Colbert et. al. 2001. The method relies on the ability of a special enzyme to detect mismatches in normal and mutant DNA strands when they are annealed. Seed was treated with ethylmethanesulphonate (EMS) to generate a population of plants with random point mutations. By selectively pooling the DNA and amplifying with fluorescently labelled primers, mismatched heteroduplexes were generated between wild type and mutant DNA. Heteroduplexes were incubated with the plant endonuclease CEL I, (cleaves heteroduplex mismatched sites) and the resultant products visualised on a ABI3730. Subsequent analysis of the individual plant DNA from the pool DNA identified the plant bearing the mutation.

 

 

The TILLING process explained…

 

TILLING diagramv1d

 

lotusmutagenisation

1: Mutagenesis

The TILLING process starts with mutagenesis to create a large population containing many 1000’s of random mutations. For TILLING in plants the customary procedure involves treating seeds with the chemical mutagen ethylmethanesulphonate (EMS) that causes G/C to A/T changes. The treated seeds are sown and the resulting M1 plants grown to produce the next generation of seeds.

 

 

 

 

lotusM2plantsB

2: Growing the TILLING population

The M1 plants are self-fertilized and the M2 seed harvested and sown. The M2 germplasm will allow recessive and lethal alleles to be recovered as heterozygotes. Each M2 plant is given a unique identifier.

 

 

 

 

lotusM2plantsA

3: DNA

DNA is individually extracted from each M2 plant and stored in 96 well plates. The M3 seeds are harvested and catalogued for future sampling.

 

 

 

 

4: Database

The unique plant identifiers allow the DNA and the archived seeds to be cross-referenced and all data is stored on a web-accessible database.

 

 

5: Pooling the DNA and amplification of Your Favourite Gene

To increase throughput the M2 DNA samples are 8x pooled. Using fluorescently labelled gene specific primers, PCR is carried out on the library of 8x pooled DNA samples. Each primer carries a different fluorescent label. In our protocol the forward strand is labelled with FAM and the reverse strand with HEX. These can then be used to identify amplification products.

PCRlarger

 

6: Heteroduplex – forming mismatched PCR fragments

In the presence of a mutant, the amplification products when heated and cooled will form mismatched heteroduplexes between the wild type and mutant DNA.

heteroduplexlarger

 

7: CEL I

To enable identification of the point mutations induced by EMS the re-annealed amplification products are incubated with a plant endonuclease called CEL I which preferentially cleaves at sites of heteroduplex mismatches that occur between wild-type and mutant DNA.

CELIlarger

 

 

8: Cleavage Products

The cleavage products are run on a capillary ABI3730 sequencer and the fluorescently labelled traces analysed. The differential end labelling of the amplification products permits the two cleavage fragments to be distinguished and pinpoints the approximate position of the mismatch.

tracea

 

9: Identification

When a mutation is detected in the pooled DNA, PCR products amplified from the individual DNA samples that make up the pool are sequenced to identify the specific plant carrying the mutation.

 

 

10: Results

The mutations are added to the database and cross-referenced with the plant line identifiers. M3 seeds from the mutant plant can then be accessed for sowing, genotyping and phenotypic analysis.