targeted induced local leisions in genome

1. TILLING
Targeting Induced Local Lesions
in Genome

2. What is Tilling
 Tilling is a non Transgenic method that helps
to identify mutations in a specific gene.
 First time this technique was developed in
Arabidopsis thaliana.
 This technique works with good results even
a population contains pre existing mutations

3. Why Tilling
 Availability of large scale sequences of most organism
 Type of Reverse Genetics
 Simple to Implement because it does not require advance
genetic tools.
 Helps to identify induced and natural variants in germplasm and
mutant collections for almost any crop species.
 Tilling has a potential to identify a large series of mutations
ranging from knockouts to subtle missense mutations
 Helps in discovering and cataloguing new polymorphisms

4. What is Mutation
 To produce change in DNA sequence
 Important breakthrough ----induction of
mutations
 Mutation causes change in genes and
phenotypes
 Forward genetics----to----Reverse Genetics
 Allowed to study higher organsim where
mutation is possible

5. Chemical Mutagen
 Alkylating agents
 yield predominantly point mutations,
 Resulting in altered and truncated protein
products
 Help to precisely map gene and protein
function

6. Chemical Mutagen----EMS
 EMS---Ethyl Methyl Sulfonate
 most commonly used chemical mutagen in
plants
 causes a high frequency of nucleotide
substitutions in a variety of organisms
 EMS alkylates guanine bases and leads to
mispairing
 alkylated G pairs with T instead of C,
resulting in primarily G/C to A/T transitions

7. Methodology
Mutation of Seeds
Chemical mutation of
seeds by using EMS
(Ethyle methanosulphonate)

8. Raising of M1 Population
 After chemical mutation
of seeds, raise M1
population.
 Self fertilize the M1
plants in order to
maintain their
homozygosity.

9. Raising of M2 Population and
collection of DNA samples
 DNA samples are
collected from M2
population.
 The samples are
pooled and arranged in
96 well plate for getting
the gene specific PCR
product.

10. Getting of PCR amplified product
 The PCR amplified
product is obtained with
the help of gene
specific primer by
using,
Automated Sequencing
Gel Apparatus

11. Denaturation and annealing of PCR
product
 The product is denatured
and allowed to reanneal by
heating and cooling.
 This will also allow the
reannealing of mutant
strand along with wild type
strand.
 The reannealing results the
formation of heteroduplexes
or polymorphism at the
point of mutation.

12. Incubation with CELI
 The double strand is
then incubated with
CELI
 CELI is a plant specific
extracellular
endonuclease
glycoprotein that
cleaves the
mismatches at the point
of heteroduplexes.

13. Generating high quality TILLING
Images
 The LI-COR 4300 DNA
Analysis System is uniquely
suited for TILLING because
it uses two-color infrared
fluorescence detection.
 With two different colors, a
true mutant has two mutant
bands
 The two color detection
method also eliminates
false positive mutations.

15. Identification of Mutant plants
The cleaved products
which are detected on
polyacrylamide
denaturing gel help to
identify the individuals
that have a mutation in
the gene of interest

18. TILLING in Major Crops
Now a days TILLING is
applied in Wheat,
Maize, Rice, Brassica
and Arabidopsis etc