A reverse genetic approach TILLING for Crop improvement

1. Targeting Induced Local
Lesions In Genome
(TILLING)

2. Targeting Induced Local Lesions In
Genome (TILLING)
Speaker
Chaudhary Ankit R.
(Reg. No. 04-AGRMA-01571-2017)
Dept. of Genetics and Plant Breeding,
C.P.C.A., S.D.A.U., Sardarkrushinagar.
Assignment
on
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3. Introduction
•Mutation can leads to change in gene or genome
expression in living organisms.
•For discovery of rare mutation in population a new
reverse genetic strategy required.
•TILLING (Targeting Induced Local Lesions In
Genome) method makes possible to discover rare
mutation at particular loci of interest in population.
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4. What is genetics???
Genetics is the science of heredity and variation in living
organisms.
Genetics
Aim :To determine gene function
Forward genetics :
Phenotype to genotype
Reverse genetics :
Genotype to phenotype
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6. What is TILLING??
 TILLING (Targeting Induced Local Lesions In
Genome) is a powerful reverse genetics approach
that uses a large mutant population for
identification of mutants in loci of interest. (Amer
et al., 2009)
 It combines chemical or physical mutagenesis and
PCR based screening to identify mutation in one or
more target gene.
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7. Historical background
Scientist Work done
Oleykowski et al., 1998
TILLING discovers mutations through the
detection of PCR products digested at mismatched
sites in heteroduplexes with Cel-1 endonuclease.
Bentley et al., 2000 First described in Drosophila.
McCallum et al., 2000
First described in Arabidopsis (Arabidopsis
thaliana).
Till et al., 2004
Perform tilling with enzymatic mixes from celery
(Apium graveolens) extracts (celery juice extracts)
[CJE].
Dong et al., 2009
Heteroduplexes can also be detected by high-
resolution melting.
Comai and Henikoff,
2006,
Gilchrist and haughn,
2010
Successfully extended to multiple model and
economic species and becoming a major tool for
functional genomics. 7

8. Concept of TILLING
Basic TILLING method allows for high-throughput identification
of single base-pair (bp) allelic variations.
Mutagenesis
Pooling of Samples
PCR
Denaturations and annealing to
form Heteroduplex
Detection of Mutations
Gel Running
Analysis
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9. Steps Involved
1. EMS mutagenesis
2. DNA preparation and pooling of individuals.
3. PCR amplification of a region of interest with
fluorescently tagged primers.
4. Denaturation and annealing to allow formation of
heteroduplexes at the site of mutation.
5. Resultant double-stranded products are digested
with CEL I, which cleaves one of the two strands
at the heteroduplex mismatches
6. Cleaved products are detected on polyacrylamide
denaturing gels. 9

10. •Seeds of selected species are mutagenized by
treatment with EMS to induce point mutation
throughout the genome.
•A founder population is grown from mutagenized
seeds i.e. Ml generation.
•Resulting Ml plants are self-fertilized to produce M2
generation.
•The M2 generation of individuals are used to
prepare DNA samples for mutational screening.
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Step-1

11. Step-2
•The DNA samples are pooled eight fold to maximize
screening efficiency (or to increase throughput).
•In order to check many samples for a possible
mutation, samples must be pooled.
•Arrayed on 96-well microtiter plates
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13. Step-3
PCR amplification of
the region of interest
in the pooled DNA
with gene specific
fluorescently tagged
primers.
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14. •The amplification products are denatured and
allowed to reanneal, generally by heating and
cooling.
•As a result, a mutant strand will often reanneal
with a wild-type strand, creating heteroduplexes at
the site of the mutation or polymorphism.
Step-4
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16. •Amplification products are incubated with an
endonuclease such as CELI or CJE (celery juice extract).
•CELI is a member of the Sl nuclease family of single
strand-specific nucleases .
•Can be overactive at 45oC and cut at large stretches of
A=T due to the looser bonds between these pairings.
•CELI cleaves the 3' side of mismatched DNA where the
heteroduplex between the wild-type and the mutant
strands of DNA loops out.
•Homoduplexes are left intact.
Steps-5
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17. •Cleavage products are
electrophoresed on automated
sequencing gel apparatus
(DNAAnalysis System).
•Gel images are analyzed with
the aid of a standard
commercial image- processing
program.
•Differential double end
labeling of amplification
products allows for rapid
visual confirmation.
Step-6
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18. •The first paper describing TILLING used to identify
mutations (McCallum et al., 2000a).
•The method was made more high throughput by
using the restriction enzyme Cel 1 combined with
the LICOR gel based system to identify mutations
(Colbert et al.,2001).
•Advantages to the LICOR system are
- Separation of large fragments
- High sample throughput (96 samples loaded on
paper combs)
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22. •The lanes that have a mutation in the pool, a band
will be visible below the wild type band on 'IR dye
700' infra red dye image.
•A counter part band will be visible in the same line
on the 'IR dye 800' infra red dye from the
complementary DNA strand.
•The sum of the length of two counterpart bands is
equal to the size of amplicon, which distinguish
mutation from amplification artifacts.
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24. Year-wise overview of TILLING approach among various crops:
Year Organism Genus species Mutagen Applied Mutagen
dose
Estimated
genome
Ploidy Mutation
rate
2000 Arabidopsis Arabidopsis
thaliana
EMS 20mM to 40 mM 125 Mbp 2X 1/300 kb
2001 Arabidopsis Arabidopsis
thaliana
EMS 20mM to 30mM 125 Mbp 2X 1000/genome
2002 Arabidopsis Arabidopsis
thaliana
Fast neutrons 60 Gy 125 Mbp 2X 0.7 to 12 kb
2003 Lotus Lotus japonicus EMS 60mL/10mL(v/v) 472Mbp 2X 1/154kb
2004 Barley Hordeum
vulgare
EMS 20mM to 30 Mm 5300 Mbp 2X 1/Mb
2005 Rice Oryza sativa MNU 1mM 430 Mbp 2X 0.80/1kb
2006 Field mustard Brassica rapa γ radiation 500Gy 500Mbp 2X 1/6190kb
2007 Pea Pisum sativum EMS 4 mM 4300 Mbp 2X 1/669 kb
2008 Soybean Glycine max EMS 50 mM 1115 Mbp 2X 1/250 kb
2009 Barley Hordeum
vulgare
Sodium azide 1.5 mM 5500 Mbp 2X 1/ 2.5 Mb
2009 Potato Solanum
tuberosum
γ radiation 0.5% to 2.0% 850Mbp 4X 1/1810 bp
2010 Field mustard Brassica rapa EMS 0-1% 500Mbp 2X 1/60kb
2010 Tomato Solanum
lycopersicum
EMS 0.7% to 1.0% 950Mbp 2X 1/322kb
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25. Organism Scientists Trait observed
Arabidopsis Tilli and Mirzabekov,
2001
Heterozygote mutations were twice folded more
than homozygote mutations
Sorghum and
Saccharum
Dillon et al., 2007 Improved for use in breeding selection
Sorghum Xin et al., 2008 Altered lignin content and increased digestibility
wheat Uauy et al., 2009 Create novel allelic diversity for breeding pasta
and common wheat
Oat Chawade et al., 2010 Increased or improved β-glucan-, antioxidant-
and omega-3 fatty acid levels, as well as
modified starch and protein content
Sunflower Sabetta et al., 2011 Downy mildew resistance
wheat Botticella et al., 2011 Decrease of their gene expression and resulted in
increased amylose content
Melons Gonzalez et al., 2011 Disease resistance and fruit quality
Tomato Okabe et al., 2011 Reduced ethylene responses
Rice Yu et al., 2012 Isolation of rice genes related to complex
quantitative traits
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26. Applications
 High through put screening of induced mutations in crops.
 Genotyping analyses to make clear the gene functions.
 For gene discovery.
 For DNA polymorphism assessment.
 Approach for functional genomics.
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27. Advantages
• Highly effective in the elucidation of gene function in
plants and animals without the production of transgenic
material.
• Detect induced mutations and naturally occurring SNPs,
as well as the detection of heterozygotes
• It produces a range of allelic mutations that are useful for
genetic analysis.
• Mutations difficult to know by forward genetics could be
revealed by TILLING.
• Detects rapidly point mutations in the population
independence of genome size, reproductive system or
generation
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28. Disadvantages
•Requires more PCR reactions.
•Rate of induction mutation is low.
•Require skillful labors.
•Increased cost for the mismatch enzyme.
•When genetic variation is high, efficiency of the
technique is decrease.
•Starting with a homozygous population is desirable.
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29. Limitations
•To obtain mutant, need to screen huge number of
individual and no guaranty that null mutant occur
•TILLING need high amount of mutagens
With low mutagens- low probability of mutant of
gene of interest and need more screening
Massive mutational- cause concomitant with gene
of interest
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