In this presentation, described in details technique about natural mutation occurs in plants ... I hope its needful to all researchers..

1. Surbhaiyya Shobha Devidas
Ph.D Scholar (Agricultural Biotechnology)
Biotechnology centre, DR. P.D.K.V, AKOLA

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TILLING (Targeting Induced Local
Lesion IN Genome)

3. TILLING (Targeting Induced Local Lesion
IN Genome)
 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
 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
Reverse genetics is-
DNA sequence Protein Phenotypes
AGCTCAATCAGATAATC
TCGAGTTAGTCTATTAG

4.  Heterozygote mutation were detected at twice the
rate of homozygote mutation
 It is the method for finding mutations produced by
chemical mutagen in specific genes
 Tilling has since been used as a reverse genetic
method in other organisms such as zebrafish,
corn,wheat, rice soyabean, and tomato etc

5. 1. Seeds (Arabidopsis) treated with chemical mutagens
(EMS, ENU)
2. Development of M1 and M2 generation
3. DNA extracted from individual M2 plant
4. Creation of DNA pool
5. PCR amplification of region of interest (Make primers
flanking gene of interest)
6. Denature DNA from pools of mutant lines
Steps in TILLING

6. 7. Allow to hybridize to wild-type DNA or formation of
heteroduplex
8. Detect mismatches in hybridized DNA
 Denaturing HPLC
 Cel I enzyme cuts at mismatches
9. Identification of the mutant individuals and
sequencing of the mutant PCR product.

7. WT
mutant
gene Z
gene Z WT
mutant
PCR amplification
from wild type
and mutant
EMS
mutagenize
seed
TILLING Work flow

8. ATGCGGACTG
|||||| |||
TACGCCGGAC
ATGCGG CTG
|||||| |||
TACGCC GAC
+
Denature DNA from
pools of mutant lines
Allow to hybridize to
wild-type DNA
Detect
mismatches in
hybridized DNA
Sequence to identify
site of mutation
DHPLC
Cel 1

9.  Mute TILLING- mutagenesis–based reverse genetic
 TechTILLING- the TILLING process
 Veggie TILLING- TILLING in vegetatively propagated plant
 In silico TILLING
 I TILLING- individualized TILLING
 Eco TILLING
Types of TILLING

10.  The Eco TILLING techniques allows polymorphism in target
genes of natural population to be quickly identified
 It facilitates the screening of gene bank collections for
desired traits
 This process was introduced by Comai.et.al (2004) using A.
thaliana and given the name eco TILLING
Eco TILLING

11.  Both natural and mutagenized populations in any
organism can be screened
 High throughput screening capacity
 99% of mutations from alkylation of guanine induced
by EMS reported as G/C to A/T transition
 Small population require to screen several mutation
 Time and saving
 No transgenic manipulations required.
Advantages of TILLING

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Marker Assisted Selection

13.  A process whereby a marker (morphological, biochemical
or one based on DNA/RNA variation) is used for indirect
selection of a genetic determinant
 Used in plant and animal breeding
 Exploits the genetic linkage between markers and important
crop traits (Edwards et al., 1987; Paterson et al., 1988)
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

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1. Reliability.
Markers should be tightly linked to target loci, preferably less
than 5 cM genetic distance.
2. DNA quantity and quality.
Some marker techniques require large amounts and high
quality of DNA, which may sometimes be difficult to obtain in
practice, and this adds to the cost of the procedures.
There are six main considerations for the use
of DNA markers in MAS:

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4. Technical procedure.
Highly simple and quick methods are highly desirable.
5. Level of polymorphism.
Ideally, the marker should be highly polymorphic in
breeding material
6. Cost.
The marker assay must be cost-effective in order for MAS
to be feasible.

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 Ideally markers should be <5 cM from a gene or QTL
Markers must be tightly-linked to target loci!
• Using a pair of flanking markers can greatly improve
reliability but increases time and cost
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM 5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%

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Markers must be polymorphic
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
RM84 RM296
P1 P2
P1 P2
Not polymorphic Polymorphic!

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F2
P2
F1
P1 x
large populations consisting of
thousands of plants
PHENOTYPIC SELECTION
Field trialsGlasshouse trials
DonorRecipient
Conventional plant breeding
Salinity screening in phytotron Bacterial blight screening
Phosphorus deficiency plot

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F2
P2
F1
P1 x
large populations consisting of
thousands of plants
ResistantSusceptible
MARKER-ASSISTED SELECTION (MAS)
Marker-assisted breeding
Method whereby phenotypic selection is based on DNA markers

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(1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
Steps involves in MAS

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P1 x F1
P1 x P2
CONVENTIONAL BACKCROSSING
BC1
VISUAL SELECTION OF BC1 PLANTS THAT
MOST CLOSELY RESEMBLE RECURRENT
PARENT
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS
THAT HAVE MOST RP MARKERS AND SMALLEST %
OF DONOR GENOME
BC2

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Advantages of MAS
 Simpler method compared to phenotypic screening
 Especially for traits with laborious screening
 May save time and resources
 Selection at seedling stage
 Important for traits such as grain quality
 Can select before transplanting in rice

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Contd…
 Increased reliability
 No environmental effects
 Can discriminate between homozygotes and
heterozygotes and select single plants
 More accurate and efficient selection of specific
genotypes
 May lead to accelerated variety development
 More efficient use of resources
 Especially field trials

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 Marker Assisted Backcrossing
 Foreground selection
 Background selection
• MAB has several advantages over conventional
backcrossing:
– Effective selection of target loci
– Minimize linkage drag
– Accelerated recovery of recurrent parent

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MAB: 1ST LEVEL OF SELECTION – FOREGROUND
SELECTION
 Selection for target gene or QTL
 Useful for traits that are difficult to evaluate
 Also useful for recessive genes
1 2 3 4
Target
locus
TARGET
LOCUS
SELECTION
FOREGROUND SELECTION
(Melchinger, 1990)

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Donor/F1 BC1
c
BC3 BC10
TARGET
LOCUS
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME
TARGET
LOCUS
LINKEDDONOR
GENES
Concept of ‘linkage drag’
• Large amounts of donor chromosome remain even after
many backcrosses
• Undesirable due to other donor genes that negatively
affect agronomic performance

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MAB: 3RD LEVEL OF SELECTION - BACKGROUND
SELECTION
 Use unlinked markers to select against
donor
 Accelerates the recovery of the recurrent
parent genome
 Savings of 2, 3 or even 4 backcross
generations may be possible
1 2 3 4
BACKGROUND
SELECTION

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Notable achievements in
MAB
 Improvement of qualitative traits
 Resistance to soybean cyst nematode
 Development of QPM genotypes
 Marker-aided pyramiding of rice genes for bacterial blight
and blast resistance
 Quantitative trait improvement
 Improvement of heterotic performance in maize
 Germplasm enhancement in tomato
 Submergence tolerance in rice cultivars
(Babu et
al.,2004)

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30. Introduction
 Normal maize - deficiency in two essential amino acids (lysine and
tryptophan) and high leucine–isoleucine ratio.
 Breakthrough came in the 1960s, discovery maize mutant opaque2
(Mertz et al., 1964)
 Encodes a transcriptional factor that regulates the expression of zein
genes and a gene encoding a ribosomal inactivating protein (Schmidt et
al., 1990)
 Reduces the level of 22-kD alpha-zeins while increasing the content of
non zein proteins particularly, EF-1 alpha, which is positively correlated
with lysine content in the endosperm (Habben et al., 1995).

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 The protein quality of opaque2 maize is 43% higher than that of common
maize(Mertz, 1992).
 opaque2 maize not popular with farmers - reduced grain yield, soft
endosperm, dull kernel appearance and susceptibility to ear rots and
stored grain pests
 QPM is a genotype in which opaque2 gene has been incorporated along
with associated modifiers.
 A genetically improved, hard endosperm quality protein maize
 Contains twice the amount of lysine and tryptophan as compared to normal maize
endosperm

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 Two additive modifier genes significantly influence the endosperm
modification in two populations viz., W64Ao2·pool 33 and pool 33·W22o2
(Lopes et al., 1995)
 One modifier locus was tightly linked with the gamma-zein coding
sequences near the centromere of chromosome 7, while the other near
the telomere of the 7L .
 Advantages of QPM
 Higher yield potential, assured seed purity, more uniform and stable
endosperm modification and less monitoring for ensuring protein quality
in seed production.

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