Spermiogenesis or Spermateleosis or metamorphosis of spermatid
TILLING and Eco-TILLING for crop improvement
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
2. 2
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
3. 3
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
8. 8
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
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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