WELCOME

Presented by :-Devidas T. Thombare

Ph. D. Scholar

Department of Plant Molecular Biology and Biotechnology, IGKV, Raipur.

TILLING (Targeted Induced Local Lesions IN Genome)

& Eco- TILLING

What is TILLING ?TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest. (McCallum et.al, 2000)

TILLING is a powerful technology that employed heteroduplex analysis to detect which organism in a population carry single nucleotide mutation in specific genes.

TILLING can also be used to detect naturally occurring SNP in genes among the accession, variety or cultivar. To study the gene function, or to detect genetic marker in population.

Reverse genetics is-

DNA sequence Protein Phenotypes

AGCTCAATCAGATA ATC

TCGAGTTAGTCTATTAG

WHY TILLING ??

Tool for functional genomics that can help decipher the functions of the thousands of newly identified genes.

To identify SNPs and/or INS/DELS in a gene of interest from population.

Genetic mutation is a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo.

Non transgenic method for reverse genetics

Discovery of TILLING

TILLING first began in the late 1990’s by McCallum who

worked on characterizing the function of two

chromomethylase gene in Arabidopsis.

Claire McCallum utilized reverse genetic approaches such as

T-DNA lines and antisense RNA, but was unable to successfully

apply these approaches to characterize CMT2.

The approach that was successful turned out to be what is now

known as TILLING.

The TILLING Methodology

Development of mutagenized population

EMS mutagenesis

Development of M2 population

DNA preparation and pooling of individuals

Mutation Discovery

PCR amplification of a region of interest

Mismatched cleavage

Detection of Heteroduplexes as extra peak or band (HPLC or

Acrylamide gel)

Identification of the mutant individual

Sequencing of Mutant PCR product.

The TILLING population - MUTAGENESIS

Mutagenizing seed with EMS done by soaking seeds in an EMS solution (14-18 h in a 30-100 mM (0.3%-1%) EMS solution.)

Planting the seeds in field

Mutagenized population (M1generation) is grown to maturity allowed to self-fertilize to produce M2 seeds.

M2 seeds can be maintained as lines or bulked

If the M2 seed is bulked then lines need to be established using M3 seed. In either case, when sampling M2 plants to establish population.

Arraying the population for TILLING

Genomic DNA is isolated from individuals M2/M3 plants

Allowing up to 8-fold pooling of diploid plants to increase TILLING throughput

Arrayed in a 96-well microtiter plate

Standardized the DNA concentration of each sample

Ethyl Methyl Sulphonate (EMS)

• Ethyl Methyl Sulphonate – Alkylating agent

• Mode of action – By transfering their alkyl group at 6-oxygen and 7-

nitrogen to the DNA base and the phosphate group.

• Produces transition mutations (G/C : A/T) because it alkylates G residues.

• Also the Ethylnitrosourea (ENU) used for induction of mutation.

CELL I Endonucleases

CEL I, isolated from celery.

S1 nuclease family of single strand-specific nucleases.

Specifically recognizes mismatches in the heteroduplex.

Cleaves DNA on the 3’ side of the mismatch (SNP).

Choosing a target sequence

The optimal length of target sequence that can be TILLED in a single

reaction is 1,500 bp. But most eukaryotic genes are over 2,000 bp and

sometimes much longer.

Since the objective in TILLING is to identify plants with deleterious

mutations in the target gene, and most mutations in non-coding sequences such

as introns, untranslated regions, and promoters will have no effect on gene

function.

So the target sequence should be chosen to minimize such sequences and

maximize coding regions.

As a first step, PCR forward and reverse primers are designed to amplify 1,500 bp or less of genomic DNA from a locus of interest.

The forward and reverse primers are differentially 5’ end labeled with IRD700 and IRD800 dye labels for fluorescent detection at ~700 nm and ~800 nm, respectively.

PCR amplification of target sequence and Heteroduplex formation

Target DNA sequence

PCR amplification

Heating and denaturing

CEL I Cut upper strand

Cut lower strand

PCR products

Homoduplexes Heteroduplexes

Denaturing electrophoresis

Heating

TILLING IN PLANTS

Arabidopsis thaliana - In 2003, Greene et al. reported that the Arabidopsis TILLING Project (ATP), which was set up and introduced as a public service for the Arabidopsis community, had detected 1,890 mutations in 192 target gene fragments.

The mutations in Arabidopsis thaliana that have been identified via TILLING have provided an allelic series of phenotypes and genotypes to elucidate gene and protein function throughout the genome for Arabidopsis researchers.

Lotus japonicus - TILLING was used to investigate induced mutations occurring in the protein kinase domain of the SYMRK gene, which is necessary for root symbiosis.

Nitrogen fixation and the functional role of sucrose synthase was the target of another Lotus japonicus TILLING study. Six isoforms of sucrose synthase were identified.

Advantages

Its applicability to virtually any organism.

Its facility for high-throughput and its independence of genome size, reproductive system or generation time.

Since it uses Chemical mutagenesis virtually all genes can be targeted by screening few individuals.

High degree of mutational saturation can be achieve without excessive collateral DNA damage.

Eco- TILLING is useful for association mapping study and linkage disequilibrium analysis.

Ecotilling can be used not only to determine the extent of variation but also to assay the level of heterozygosity within a gene.

Source: Muhammad Rashid et al, 2011

• The first publication of the EcoTILLING method in which TILLING was modified to mine for natural polymorphisms was in 2004 from work in Arabidopsis thaliana.

• EcoTILLING is similar to TILLING, except that its objective is to identify natural genetic variation as opposed to induced mutations.

• Many species are not amenable to chemical mutagenesis; therefore, EcoTILLING can aid in the discovery of natural variants and their putative gene function

•This approach allows one to rapidly screen through many samples with a gene of interest to identify naturally occurring SNPs and / or small INs/DELS.

EcoTILLING

TILLING and EcoTILLING

Cases study

Results

We demonstrate that high throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse genetic resources currently available.

We screen the pools of DNA sample for mutation in 1-kb segment from 11 different gens, obtaining 17 independent induced mutations from a population of 750 pollen mutagenized maize plants.

One of the gene targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutation that are predicted be strongly deleterious.