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Assignment -2
TILLING AND ECOTILLING IN CROP IMPROVEMENTS
DEPARTMENT OF AGRICULTURAL BOTANY

Course Teacher : Dr. Mangesh Mohril,

Professor, Post Graduate Institute, Biotechnology Centre,
Department of Agricultural Botany, Dr. PDKV,Akola

Presented by,
Mr. Tahakik Rushikesh Ravindra
PhD Research Scholar, Dr. PDKV,Akola
 CONTENT

1. INTRODUCTION

2. DISCOVERY OF TILLING AND EcoTILLING ?

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3. Methodology

4. Why Eco-TILLING

5. Still We Need TILLING

6. Merits and Demerits

7. Applications

2
Variation in nucleotide sequence is mainly determine heritability of phenotypic
differences and has been exploited by human for the improvement of crops
since the starting of domestication.
 INTRODUCTION
• TILLING (Targeting Induced Local Lesions IN Genomes) mutation detection tool. It is a non-
transgenic reverse genetic technique that is suitable for most plants.
• For decades, mutations are created by treatment with the application of the same chemical
mutagens have been employed for TILLING successfully.
• TILLING could give mutations that are missense in allelic series and truncation through
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inducing primarily random point mutations at high density using chemical mutagens.
• EcoTIILING, an expansion of TILLING technique, can be used to discover point mutations or
polymorphisms in natural populations
• The difference between two techniques stated that, TILLING relies or depends on chemical
methods to induce
Reverse genetics is- mutations in a given plant population and Eco TILLING uses various
advanced technologies to find and use desirable gene versions or variants in the naturally
DNA sequence
mutated population. Protein Phenotypes

• TILLING and Eco TILLING

AGCTCAATCAGATA ATC also can be identify unknown and known point mutations from a
set of candidate genes both methodologies provide proof of function for both natural and induced
TCGAGTTAGTCTATTAG
variations.
 DISCOVERY OF TILLING AND EcoTILLING

· TILLING is mutation detection tool, that offers potential to study gene function at functional level.
· TILLING was first developed for Arabidopsis as a novel reverse genetic technique (Clair McCallum
et al. 2000)
· It is a method in molecular biology that allows direct identification of mutation in a specific gene
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· It is reverse genetic method that combines chemical mutagenesis (EMS, ENU) with high throughput
genome-wide screening for point mutation detection in gene of interest.
• TILLING identifies induced mutations in mutagenized populations, However modified form known
as Eco TILLING detects naturally occurring SNPs, especially in landraces and wild accessions.
• TILLING has proved to have additional benefits in addition to identifying polymorphism in genomes.
The first part of TILLING requires the development of large number of mutagenized populations.
• TILLING directly introduces genetic variation on improved or elite germplasm, it avoids the need for
introgression of a mutant allele in a no adapted background into current high-yielding varieties and
avoids the problem of linkage drag.
 TILLING FORMATION PROCEDURE
DEPARTMENT OF AGRICULTURAL BOTANY
• Chemical mutagenesis is
utilized.
• Chemical agents include EMS
(ethyl methanesulfonate).
• Treatment of seeds with EMS
(20-40 mM) for 10-20 hours.
• F1 plants obtained by growing
treated seeds.
Creating Mutated
Populations:
Development of
Mutant
Generations:
• F1 plants self-fertilized to
produce F2 lines.
• M1 and M2 generations
developed.
• M3 seed bank established.
• PCR amplification of targeted
DNA segment using pooled
DNA, gene region of choice
amplify using SNP primer
tagged with IRD700 and
IRD800 fluorescence dyes.
• Then PCR products are
reanneal and under condition
that single base mismatch are
prevented to hybridize using
CEL-1(specifically cut single
base mismatch)
Detection of
Mutations in
Targeted Sequence:
Analysis of Mutant
Phenotype:
• Then PCR products are
reanneal and under condition
that single base mismatch are
prevented to hybridize using
CEL-1(specifically cut single
base mismatch)
• PCR fragments are analysed by
using LiCor sequencing gel.
• Data stored in databases for
future reference.
 Eco-TILLING FORMATION PROCEDURE
Selection of Target Genes:
• Identify genes of interest for mutation screening based on their relevance to the desired traits or
phenotypes.
• Prioritize genes involved in ecological adaptation, stress response, or other important biological
processes.

Population Collection:
• Obtain a diverse population of individuals from the target species across different ecological niches
or geographical locations.
• Ensure representation of genetic variation within the population.
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DNA Extraction:
Homoduplex
• Isolate genomic DNA from individual plants within the population.
• Ensure high-quality DNA extraction to facilitate accurate mutation screening.
Hetroduplex
Pooling of DNA Samples:
• Combine DNA samples from multiple individuals into pools (typically 5-8 plants per pool).
• Minimize costs and streamline screening process by reducing the number of samples to be Homoduplex
analyzed individually.
Hetroduplex
PCR Amplification:
• Design SNP primers targeting the region of interest within the gene.
• Perform PCR amplification using pooled DNA samples as templates.

Mutation Detection:
• Utilize mismatch-specific endonucleases or other mutation detection methods (e.g., DHPLC, high-
throughput sequencing) to identify naturally occurring mutations within the PCR-amplified DNA
fragments.
• Screen for alterations in DNA sequence indicative of mutations.
 APPLICATION OF TILLING & EcoTILLING
Application of TILLING in Plants:
• Non-Transgenic Approach:
• Avoids the introduction of foreign genes into the genome,
maintaining natural genetic composition.
• High Throughput:
• Enables screening of large populations for mutations efficiently,
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facilitating rapid genetic analysis.
• Precise Mutation Localization:
• Provides approximate location of induced mutations within a few
base pairs, allowing targeted sequencing.
• Mutational Diversity:
• Chemical mutagenesis produces various mutations (nonsense,
splice site, missense), offering insights into gene function.
• Time and Cost Efficiency:
• Saves time and money by not requiring sequencing of all
individuals in a population to identify mutations.
• Sensitive Detection:
• Sensitive enough to detect induced mutations, naturally occurring
SNPs, and heterozygotes.
Application of EcoTILLING in Plants:
• Natural Variation Detection:
• Uncovers natural genetic variation without the need for
mutagenesis, providing insights into population diversity.
• Rapid Screening:
• Rapidly screens large populations to identify naturally occurring
SNPs and DNA polymorphisms.
• Suitable for Non-Amenable Species:
• Applicable to species not suitable for chemical mutagenesis,
expanding the range of target organisms.
• Role in Resistance Gene Mining:
• Identifies variation in resistance genes to speed up immunity
identification against diseases.
• Successful application in barley's mlo and Mla resistance genes
against powdery mildew.
• Mutation Frequency Determination:
• Provides a direct measure of induced mutations compared to
conventional mutation breeding.
• Allows for the selection of numerous genes in parallel and
forecasting of recognized alleles based on mutation frequency and
library size.
 LIMITATION OF TILLING & Eco TILLING

Limitation of TILLING in Plants : Limitation of Eco TILLING in Plants:

• Off-Target Effects: • Limited to Natural Variation:
• Chemical mutagenesis can induce unintended mutations outside of • Restricted to identifying existing genetic variation and may not
the target gene region, potentially leading to unpredictable uncover novel mutations.
phenotypic effects. • Complexity in Heterozygous Species:
• Labor-Intensive Screening: • Challenges in distinguishing heterozygous genotypes, especially in
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• Screening large populations for mutations can be labor-intensive
and time-consuming, requiring careful analysis of numerous
samples.
• Limited to Point Mutations:
• TILLING primarily detects point mutations, limiting its ability to
capture larger structural variations or gene duplications/deletions.
• Biased Mutation Spectrum:
• Chemical mutagens may induce mutations biased towards certain
types (e.g., transitions vs. transversions), potentially skewing
genetic analysis results.
• Risk of Undesired Traits:
• Mutations induced by chemical mutagens may inadvertently
introduce undesirable traits, complicating the interpretation of
phenotypic data
highly heterozygous species.
• Limited to Known Variation:
• EcoTILLING relies on the existing genetic variation within a
population, which may limit its ability to identify novel mutations or
rare alleles.
• Challenges in Data Interpretation:
• Analyzing natural genetic variation can be complex, especially in
diverse populations where distinguishing between background
variation and functional mutations can be challenging.
• Dependency on Genetic Diversity:
• The effectiveness of EcoTILLING is highly dependent on the genetic
diversity within the studied population, which may vary among
species and populations.

• Genetic variation is
fundamental in determining
differences in traits
inherited by organisms.
• It has been harnessed by
humans for crop
improvement since the
dawn of domestication.
Role of Genetic
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Variation in Phenotypic
Diversity:
CONCLUSION
• Variation can arise naturally
from diverse populations or
be induced through
mutagen treatment.
• Mutations induced by
chemical mutagens have
been widely employed for
TILLING.
Natural and Induced
Variation:
• Chemical mutagens,
particularly chemical
mutagenesis, are potent
tools for reverse genetics in
plants.
Utility of Chemical
Mutagenesis:
• TILLING is a non-transgenic • Facilitate genetic mapping
reverse genetic method through linkage analysis by
used across various plant identifying variation.
species. • Allow for precise detection
• EcoTILLING, an extension of of mutations, even in
TILLING, explores natural populations with existing
genetic variation within genetic variations.
populations.
TILLING and Benefits of TILLING and
EcoTILLING Techniques: EcoTILLING:
• TILLING and EcoTILLING
continue to be crucial in
genetic research and crop
improvement.
• Advancements in
technology will further
enhance their utility in plant
molecular biology.
Adaptable to diverse
plant species,
irrespective of size or
genetic complexity.
DEPARTMENT OF AGRICULTURAL BOTANY