Provide basics of TILLING approach for identification of point mutations in various breeding populations and utilize these mutations.

1. Detection of Single Nucleotide Polymorphism
- TILLING and Eco-TILLING
COURSE – GP-604
Presented by Presented to
Raju Ram Choudhary Dr. Mukesh Kumar
Adm. No. 2019A48D
Assignment on

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What is SNP ?
All SNP are product of
mutation but all
mutations are not SNPs
Novel SNPs Discovery Methods
 DNA sequencing
 Capillary Electrophoresis
 Mass Spectrometry
 Single-strand Conformation Polymorphism (SSCP)
 Single-base Extension;
 Electrochemical Analysis;
 Denaturing Gradient Gel Electrophoresis
 TILLING and its variants

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 Stands for Targeted Induced Local Lesions IN Genome
 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
 Takes advantage of classical mutagenesis, sequence availability and high-
throughput screening for nucleotide polymorphisms in a targeted sequence
Reverse genetics is-
What is TILLING?

4. Cont…
 Tool for combine high density of point mutations provided
by traditional chemical mutagenesis with rapid mutational
screening to discover induced lesions
 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 technique for gene modification

5. Why TILLING is preferred over other Reverse Genetics
approaches
 It can be applied to any plant species, regardless of its genome
size, ploidy level or method of propagation
 It is recommended as non-GMO technology, so when using
TILLING, GMO procedures and controversies are avoided
 TILLING does not require transformation and, thus, is the only
reverse genetics strategy applicable for species that are not
transformable or recalcitrant.
 In crop breeding as an alternative to the transgenic approach

6.  TILLING first began in the year, 2000 by McCallum et al.
who worked on characterizing the function of 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.
 Modification of TILLING protocol by Colbert et al. (2001) –
Instead of DHPLC, Use a mismatch specific celery nuclease,
CEL1 to identify SNPs
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Discovery of TILLING

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Overview

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 Step-1 Creation of mutated populations
 Chemical mutagenesis
 Development of M1 and M2 generations
 DNA extraction from individual M2 plants
 Creation of DNA pools of 5-8 M2 plants
 Setting up an M3 seed bank
 Step-2 Detection of mutations in a targeted sequence
 PCR amplification of the targeted DNA segment using pooled DNA as a
template
 Detection of mutations using different procedures, e. g. cleavage by
specific endonuclease, DHPLC and high-throughput sequencing
 Identification of the individual M2 plant carrying the mutation
 Sequencing the target gene segment to confirm the mutation and to
determine the type of nucleotide change
 Step-3. Analysis of the mutant phenotype
TILLING platform in plants includes the following steps

9. Chemical mutagenesis
 The high density (1 mutation/ 300 kb) of chemically induced point
mutations makes TILLING suitable for targeting small genes
 EMS is the mutagen used most often
 Why EMS?
 EMS alkylates guanine bases and leads to mispairing, alkylated
guanine pairs with thymine results in G/C to A/T transitions
 It induces large number of recessive mutations
 High degree of mutational saturation can be achieved without
excessive DNA damage
 Stable mutagenesis
 Other mutagen - N-methyl-N-nitrosourea and Sodium azide
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10. Pooling of Samples
An individual plate has 64 wells in
use, each with DNA from a single
unique individual
The Pool plate takes the individual
DNA samples from a whole column of
an individual plate and puts it into one
well. A total of 12 individual plates
are pooled this way
 Everything is carefully marked so that if a mutation is detected,
the individual plate and column are known
After pooling, PCR begins...

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 Primers must be carefully selected to ensure that they are going
to amplify a suitable region
 don’t want to amplify non-coding region
 use of a longer primer and high Tm helps to increase
specificity, and decrease noise on the LI-COR gel
 About 45 cycles are required to reach 10ng/ul level
 End step of PCR is to denature all DNA present, then re-anneal
 This causes a small bubble to form between mismatched pairs
of DNA (where the mutation has occurred) forming a
heteroduplex
 Labelling of primers with 2 different dyes occurs in order to
facilitate imaging detection process
PCR

12. Heteroduplex Formation
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13. Detection of mutations
 DHPLC (Denaturing High Performance Liquid Chromatography)
This is the method used originally, but now the enzyme Cel-1 is used
Not as useful for high throughput because of the time required to run a
sample
Can detect heteroduplexes with good efficiency, but cannot give good
specificity as to where the mutation is in the gene
 Cel-1 (celery nuclease)
derived from celery
Member of S1 nucleases family of SS-specific nucleases
cuts DNA at a mismatch (heteroduplex)
exact role in cell is not known but may function to cut up single
stranded nucleic acids from infecting viruses
cuts at 3’ end of mismatch
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14. Cel-1 is added to the final PCR products and cuts at
bubbles formed in heteroduplexes
After digestion, reaction is stopped
Sephadex beads are used to clean up each sample so
that only water and DNA are left
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17. LI-COR Gel Running
 Samples are loaded onto a comb using either a robot or manually
with a pipettor
 Comb is used to load samples onto a LI-COR Gel
 Samples are run until they run completely off the gel
 LI-COR gel running machine detects fluorescent tags on
fragments and creates a real time image of the gel as it runs.
 Since each fragment should be labelled with the 2 different dyes
used, if there is a mismatch and the DNA is cut, two smaller
fragments will be present, one labelled green, one red
 Through this methodology, an almost exact identification of the
base pair where the mutation occurred is possible
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18. Analysis
 The sum of the length of two counterpart bands is equal to the
size of amplicon, which distinguish mutation from
amplification artifacts.
 After finding a mutation, the mutation can be narrowed down
the almost the exact base pair, but it could be one of 8 different
individuals because of the pooling process
 The individual plate where the pooled samples came from is re
run with the eventual idea being that each individual gets its
own lane on the gel
 This allows for exact identification of the individual that
carries the mutation
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19. Results of TILLING
• Allelic Series Created
• Due to different mutations causing either truncations,
single amino acid changes, etc, mutations affecting the
protein of interest are varied
– this allows for an allelic series which may cause differing
phenotypes and allow for greater understanding of protein
function than a single knockout could provide
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20. Merits of TILLING
 It is independent of genome size, reproductive system or
generation time.
 High throughput & data analysis can also be automated.
 Valuable for essential genes, where sublethal alleles are
required for phenotypic analysis.
 TILLING is suitable for any organism that can be heavily
mutagenized, even those that lack genetic tools.
 Organisms that do not have efficient transformation systems –
TILLING is the only practical choice
 TILLING can introduce genetic variation in an elite
germplasm without need to acquire variation & thus avoiding
introduction of agriculturally undesirable traits.
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21. Cont…
 Also overcomes problems of transgenic approach as it is
independent of transgene efficiency and regeneration of plant.
 The likelihood of recovering a deleterious mutation can be
calculated in advance.
 Highly sensitive and cost effective Detect SNP over thousands
of samples.
 Producing phenotypic variants without introducing foreign
DNA of any type into a plant’s genome
 Has the advantage of exemption from regulatory approval
requirements which is strictly obliged for transgenic crops
when commercially suitable variations are discovered.
 No requirement of sophisticated tissue culture methodology
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22. • 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.
Eco-TILLING

23. Eco-TILLING Applications
• Mapping- Detection of polymorphisms that can be used as
genetic markers
• Association analysis- Correlating natural polymorphisms with
phenotypic traits
• Mutational profiling- Characterizing the amount of genetic
variation in a species
• Biodiversity- Determining the evolutionary history of natural
populations
• Eco- TILLING is useful for association mapping study and
linkage disequilibrium analysis.
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25. SYMBOL OF TRUST