This case study describes the discovery of induced point mutations in maize genes using the TILLING technique. Researchers screened a population of 750 pollen-mutagenized maize plants for mutations in 11 genes. They detected 17 independent mutations in total, including an allelic series of 3 mutations in the DMT102 gene. No mutations were found in 5 other genes screened. The study demonstrates the ability of TILLING to discover mutations and further characterize gene function in crops like maize.

2. PRESENTED BY :
JADHAV R. A
REG.NO. 2015A06P

3. POINTS TO BE COVERED
Introduction
Forward & Reverse genetics
What is TILLING & EcoTILLING?
Principles of TILLING
Steps involved
Mutant generation for TILLING
TILLING centres
TILLING in plants
Advantages & Applications
Case Studies

4. INTRODUCTION
 Genetic variation exists naturally or may be generated by
mutagenesis techniques.
 Genetic variation associated with phenotypic change.
 The most common form of genetic variation is due to single
nucleotide change, insertion and deletion, which generates
single nucleotide polymorphisms (SNPs).
 Reverse genetics techniques allow recognition of possible loss of
function for a particular gene during early stages of development.
(Stemple 2004)

5. FORWARD VS. REVERSE GENETICS
Forward Genetics Reverse Genetics
Phenotype Genotype Genotype Phenotype
1. Select a biological process 1. Select a gene or genes of interest
2. Generate mutant populations 2. Generate mutant populations
3. Screen for mutants with a desired
phenotype
3.Develop and conduct sequence-
based mutant screens
4. Map and clone the gene
responsible for the phenotype.
4.Analyze the phenotype of the
mutants

7.  Targeting Induced Local Lesions IN Genome (TILLING)
 “A method in molecular biology that allows directed identification
of mutations in a specific gene”.
 Introduced in 2000, using the model plant Arabidopsis thaliana by
McCallum who worked on characterizing the function of two
chromo methylase gene in Arabidopsis.
 It is a powerful technique which identifies single base changes in a
specific gene in a mutagenized population
(McCallum et al. 2000)
 Used in corn, wheat, rice, soybean, tomato and lettuce
WHAT IS TILLING ?

8.  It employed heteroduplex analysis to detect which organism
in a population carry single nucleotide mutation in specific
genes.
 These techniques allow identifying diverse versions of genes
in a germplasm and acquiring information about their gene
function. This information can provide guidelines to develop
new strategies for genetic improvement of plants.
 An expansion of the TILLING technique is Eco TILLING,
which can be used to discover point mutations or
polymorphisms in natural populations. (Comai et al. 2004).
 Both can be used to identify unknown and known point
mutations from a set of candidate genes.

9. PRINCIPLE OF TILLING
 The method combines a standard and efficient technique of
mutagenesis using a chemical mutagen such as EMS with a
sensitive DNA screening technique that identifies single base
mutations (point mutations) in a target gene.
 Based on the formation of DNA heteroduplexes that are
formed when multiple alleles are amplified by PCR and are
then heated and slowly cooled.
 A “bubble” forms at the mismatch of the two DNA strands,
which is then cleaved by a single stranded nucleases.
 The products are then separated by size on several different
platforms.
 Mismatches may be due to induced mutation, heterozygosity
within an individual, or natural variation between individuals.

11. STEPS INVOLVED
 EMS mutagenesis
 DNA preparation & pooling of individuals
 PCR amplification of a region of interest with fluorescently
tagged primers
 Denaturation & annealing to allow formation of
heteroduplexes at the site of mutation
 Resultant double stranded products are digested with CEL I,
which cleaves one of the two strands at the heteroduplex
mismatches
 Cleaved products are detected on Polyacrylamide denaturing
gels.
(Till et al. 2003).

13. STEP 1
 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.
M1 generation
 Resulting M1 plants are self fertilized to produce M2
generation
 The M2 generation of individuals are used to prepare DNA
samples for mutational screening.

15. CEL I ENDONUCLEASES
 CEL I, isolated from celery.
 S1 nuclease family of single strand-specific
nucleases.
 Specifically recognizes mismatches in the
heteroduplex
 Cleaves DNA on the 3’ side of the mismatch (SNP).

16. STEP 2
 The DNA samples are pooled eight fold to maximize
screening efficiency
 In order to check many samples for a possible
mutation, samples must be pooled
 Arrayed on 96-well microtiter plates
 Genomic DNA isolated from mutagenized samples can
be pooled with up to eight individuals and arrayed in
micro titer plates.

17.  Equal amounts of DNA from each individual sample. If the
concentration of any DNA sample is lower than others,
mutations within the pool might not be detected.
 The pooled DNA samples are amplified by PCR using gene
specific primers which are designed to amplify 1-1.5 kilobase
(kb) fragments and are end labeled with two different
fluorescent dyes.
 The amplified PCR products are denatured and re-natured by
heating and cooling to form a heteroduplex.
(Till et al. 2003).

18.  If the pooled DNA contains a single nucleotide mutation in
the target sequence, the heteroduplex between wild and
mutant alleles is cleaved precisely at the 3’ end of the
mismatch Endonuclease Cel I, leaving the wild type
duplex intact.
 The cleaved products can be separated easily by either 10%
polyacrylamide gel followed by staining with SYBR Green
or the most commonly used apparatus LI-COR IR2 gel
analyzer (Colbert et al.)

19. MUTANT GENERATION FOR TILLING
 The most commonly used mutagenic agents: Ethyl methyl
sulfonate (EMS) and N-ethyl-N-nitrosourea
(ENU);deaminating agents, including Nitrous and Nitroso
guanidine; hydroxylating agents, including hydroxylamine .
 EMS produces random mutations by nucleotide substitution;
specifically by guanine alkylation. This typically produces
only point mutations in the DNA sequence.
 This mutagen induces a large number of recessive mutations
in plants.
 Soaking of seeds in 20-40 millimolar (mM) EMS for 10- 20
hrs. and grew them to obtain M1 plants. Since M1 plants are
chimeric for mutations, they self fertilized M1s to produce
M2 lines for DNA isolation.

20. TILLING CENTRES
Several TILLING centers exist over the world
 Rice – UC Davis (USA)
 Maize – Purdue University (USA)
 Brassica napus – University of British, Columbia (CA)
 Brassica rapa – John Innes Centre (UK)
 Arabidopsis – Fred Hutchinson Cancer Research
 Soybean – Southern Illinois University (USA)
 Lotus and Medicago – John Innes Centre (UK)
 Wheat – UC Davis (USA)
 Pea, Tomato - INRA (France)
 Tomato -University of Hyderabad (India)

21. TILLING IN PLANTS
Arabidopsis thaliana :
 In 2003, Greene et al. reported that the Arabidopsis
TILLING Project (ATP), which was set up and introduced as
a public service for the Arabidopsis community, had detected
1,890 mutations in 192 target gene fragments.
 The mutations in Arabidopsis thaliana that have been
identified via TILLING have provided an allelic series of
phenotypes and genotypes to elucidate gene and protein
function throughout the genome.

22. WHEAT
 Slade et al (2005) studied the Granule Bound Starch
Synthase 1 (GBSSI) or waxy starch gene.
 With only one or two functional GBSSI genes wheat produces
starch with intermediate levels of amylopectin.
 Amylopectin has economically valuable functional qualities
and unique physio-chemical properties.
 They identified a total of 246 novel alleles of waxy genes out
of 1920 EMS mutagenized lines from hexa- and tetraploid
wheat.
 84 missense mutations were found among 246 new alleles and
54 of the 84 mutations were predicted to severely affect protein
function

23. RICE
 Small genome of about 430Mb (Mega bases).
 There are two major subspecies of cultivated rice, Oryza
sativa ssp. japonica and ssp. indica. The rice genome has
been sequenced and was estimated to contain 50, 000 genes.
 Wu et al. (2005) screened 2000 individuals from a population
mutated with 0.8% and 1.0% EMS.
 From 10 genes, they detected two independent mutations in
two genes, pp2A4 encoding a serine/threonine protein
phosphatase catalytic subunit, and C7 encoding a callose
synthase, with a mutation rate of 0.5 mutations per Mb.
 A 1.6% EMS concentration generated the highest frequency
of mutations.
 However, albino (8.7%) was the most frequently observed
phenotype.

24. ADVANTAGES
 Its applicability to any organism.
 Its facility for high-throughput and its independence of
genome size, reproductive system or generation time.
 Since it uses chemical mutagenesis virtually all genes can be
targeted by screening few individuals
 High degree of mutational saturation can be achieved
without excessive collateral DNA damage
 Non transgenic method for reverse genetics.
 It acts as a tool for functional genomics that can help
decipher the functions of the thousands of newly identified
genes.
 It is useful to identify SNPs and/or INS/DELS in a gene of
interest from population.

25.  Genetic mutation is a powerful tool that establishes a direct
link between the biochemical function of a gene product and
its role in vivo.
 Eco-TILLING is useful for association mapping study and
linkage disequilibrium analysis.
 Eco-tilling can be used not only to determine the extent of
variation but also to assay the level of heterozygosity within a
gene

27. APPLICATION OF TILLING
 For gene discovery
 CODDLE (Codons Optimized to Detect Deleterious Lesions) was
developed as a general tool for polymorphism analysis and for
designing of primers for any organism and for detection of any
mutation based on DNA.
 For TILLING and for polymorphism analysis, there is a need to
assess the effect of missense mutations.
 For example, when specific DNA sequence with the length of 1 kb was
selected,
 CODDLE can evaluate whether a missense mutation is likely to have an
effect on the encoded protein.

28. FOR DNA POLYMORPHISM ASSESSMENT
 It plays an important role in biological evolution.
 The methods currently available for revealing DNA polymorphism
includes DNA sequencing, single-strand conformation
polymorphism (SSCP), hybridization, and microarray, and these
methods have their own advantages and limitations.
 Although DNA sequencing is simple and straight-forward, it is rather
costly and time-consuming. SSCP provides a high-throughout
strategy for polymorphism detection; however, it has low efficiency in
detecting novel mutations with a limit of 200 to 300 bp length of target
DNA sequence.
 Microarray holds two disadvantages, one is high cost of operation,
and the other is the low detecting-frequency of less than 50%.
 Based on TILLING, a strategy, referred to as ecotypic TILLING
(EcoTILLING), was developed to detect DNA polymorphism present
in naturally occurring mutations

29. FOR CROP BREEDING
 Unlike conventional mutation breeding in which the
mutation frequency is unknown or estimated only from
mutations conveying a visible phenotype, TILLING
provides a direct measure of induced mutations.
 Furthermore, TILLING allows not only the rapid, parallel
screening of several genes but also a prediction of the
number of alleles that will be identified on the basis of
the mutation frequency and library size.

30. ECOTILLING

33.  EcoTILLING is a method that uses TILLING techniques to
look for natural mutations in individuals, usually for
population genetics analysis.
 DEcoTILLING is a modification of TILLING and
EcoTILLING which uses an inexpensive method to identify
fragments.
 Since the advent of NGS sequencing technologies, TILLING
by sequencing has been developed based on Illumina
sequencing of target genes amplified from multi-
dimensionally pooled templates to identify possible single
nucleotide changes.

34.  EcoTILLING can detect DNA variations from single nucleotide
polymorphism (SNP), small fragment insertion and deletions to
simple sequence repeat (SSR).
 EcoTILLING only the sequencing of the unique haplotypes is
required to determine the exact nucleotide polymorphism at a locus,
 By using Ecotilling, Comai et al discovered 55 haplotypes from 150
individual plants in five genes, whose sequences ranged from those
differing by a single-nucleotide polymorphism to those representing
complex haplotypes.

36. CASE STUDY 1
 BMC Plant Biology
 Methodology article Open Access
 Discovery of induced point
mutations in maize genes by
TILLING
 Bradley J Till1, Steven H Reynolds2, Clifford Weil3, Nathan Springer4,
Chris Burtner2, Kim Young1, Elisabeth Bowers1, Christine A Codomo1,
Linda C Enns2, Anthony R Odden1, Elizabeth A Greene1, Luca Comai2
and Steven Henikoff*1
 Address: 1Basic Sciences Division, Fred Hutchinson Cancer Research
Center, Seattle, Washington 98109, USA, 2Department of Biology,
University of Washington, Seattle, Washington 98195, USA, 3Department
of Agronomy, Purdue University, West Lafayette, Indiana 47907, USA
and 4Department of Plant Biology, University of Minnesota, St. Paul,
Minnesota, 55108 USA

37. MATERIALS & METHODS
 Fresh pollen is collected and mutagenized with EMS.
 Pollen is then applied to silks of wild-type plants from the same genetic
background. Seeds from the resulting ears are grown into plants of the M1
generation.
 Tissue is collected either from each M1 plant or from approx. 10 M2
siblings from the M1 self cross. M3 seed is generated by randomly
intermating 10– 12 M2 siblings. This M3 seed serves as the seed stock for
future studies.
 DNA is extracted from collected tissue and samples are pooled to increase
screening throughput. For mutation detection, sequence specific primers are
used to amplify the target locus by PCR.
 Heteroduplexes are cleaved using CEL I endonuclease and are visualized
using denaturing polyacrylamide gel electrophoresis.

39. MUTATIONS DETECTED BY SCREENING EMS-MUTAGENIZED MAIZE POPULATIONS
Gene Individual Sequence Stock number
DMT101 3D3 C553T UI20118
8A8 G464A UI20291
DMT102 3H3 C677T UI20122
11B3 G878A UI20424
12B3 G878A UI20459
15G3 G938A NS2809.1
19C2 G676A NS3457.3
21G2 A637W NS3471.92
DMT103 4A9 G116A UI20152
14E9 G514A NS2806.7
16G9 G519A NS2810.4
DMT106 15E2 C697T NS2808.6
HAC110 16B7 G471A NS2809.12
HDA105 4A3 G343A UI20152
9F2 C571T UI20333

40. RESULTS
 They screened pools of DNA samples for mutations in 1-kb
segments from 11 different genes, obtaining 17 independent
induced mutations from a population of 750 pollen-
mutagenized maize plants.
 One of the genes targeted was the DMT102
chromomethylase gene, for which they obtained an allelic
series of three missense mutations that are predicted to be
strongly deleterious.
 No mutations were detected in 1 kb fragments of five other
genes screened: DMT105, SDG104, SDG105, SDG124 and
SDG125.
 Multiple non-G/C-to-A/T mutations discovered in NS3471.9
indicate that it derives from contaminating pollen.

41.  Development and Characterization of a New
TILLING Population of Common Bread Wheat
(Triticum aestivum L.)
 Liang Chen1 , Linzhou Huang1 , Donghong Min1 , Andy
Phillips2, Shiqiang Wang1 , Pippa J. Madgwick2 , Martin A. J.
Parry2 , Yin-Gang Hu1,3*
 1 State Key Laboratory of Crop Stress Biology in Arid Areas and
College of Agronomy, Northwest Agricultural and Forestry
University, Yangling, Shaanxi, China, 2 Centre for Crop Genetic
Improvement, Department of Plant Science, Rothamsted Research,
Harpenden, Herts, United Kingdom, 3 Institute of Water Saving
Agriculture in Arid Regions of China, Northwest Agricultural and
Forestry University, Yangling, Shaanxi, China
CASE STUDY 2

42.  They developed a new TILLING resource comprising 2610 M2 mutants
in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with
altered morphological and agronomic traits were observed from the M2
and M3 lines in the field.
 The value of this new resource was tested using PCR with RAPD and
Intron-spliced junction (ISJ) primers, and also TILLING in three selected
candidate genes, in 300 and 512 mutant lines, revealing high mutation
densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING.
 In total, 31 novel alleles were identified in the 3 targeted genes and
confirmed by sequencing.
 The results indicate that this mutant population represents a useful
resource for the wheat research community. The use of this reverse
genetics resource will provide novel allelic diversity for wheat
improvement and functional genomics.

43. MATERIALS AND METHODS
 Mutagenesis and plant growth conditions
 A Chinese common wheat cultivar Jinmai47, with higher
drought-tolerance and good agronomic traits.
 3000 seeds were soaked in 0.8% (v/v) EMS solution at the
ratio, 350 grains/100 mL with gentle agitation overnight (18
h) at room temperature.
 After EMS treatment, seeds were thoroughly washed with tap
water for 3 hours.
 The EMS-treated seeds (M1) were sown in pots with soil in a
greenhouse until harvest. M2 seeds were harvested from 1350
M1 individuals, threshed and packed.

44. DNA ISOLATION AND PREPARATION OF DNA
POOLING
 Genomic DNA from young leaves was isolated from 2610
M2 plants using the CTAB method.
 DNA concentrations were measured using a
Spectrophotometer and normalized to 50 ng/ mL. Then DNA
samples from 4 M2 plants with equivalent amounts were
pooled for initial screening.
 PCR with RAPD :
 The six RAPD markers for estimating mutation frequency are

45. PRIMER SEQUENCES OF THE 6 PRIMERS USED
AND THE MUTATION FREQUENCY DETECTED
Prime
r
Sequence
(59- 39)
Primer
length
No. of
bands
in wild
type
No. of
mutati
on
bands
M2
plants
screene
d
Mutation
Freq.
A-09 GGGTAACGCC 10 6 1 300 1/36 kb
A-10 GTGATCGCAG 10 8 3 300 1/16 kb
UBC3 CCTGGGCTTA 10 7 4 300 1/10 kb
R1 TCGTGGCTGAC
TTCACTG
18 8 1 300 1/86 kb
E4 GAATTCCAGCC
TGCA
15 7 2 300 1/31 kb
IT31 GAAGCCGCAG
GTAAG
15 6 2 300 1/27 kb

46. PHENOTYPIC ANALYSIS
 Sequence analysis was carried out to determine the effects of
mutations based on the probability of affecting protein function.
 The PARSESNP (Project Aligned Related Sequences and Evaluate
SNPs) and SIFT (Sorting Intolerant From Tolerant) programs were
used to predict the severity of mutation identified.
 To verify the mutation effect of mutant, photoperiod gene identified
by TILLING, and examine the agronomic traits of some mutants,
mutant lines were advanced to the M4 or M5 generation
 The heading date and flowering time were also recorded.

47. RESULTS

48. FREQUENCY OF TYPICAL MUTATIONS OBSERVED AMONG THE
2,610 M2 INDIVIDUALS SCREENED
Phenotype description Number of mutants
observed
Frequency
Dwarf and semi-dwarf 35 1.34%
Spike morphology 16 0.61%
Albinism 7 0.27%
Late heading 7 0.27%
Lower fertility 5 0.19%
Few tillers 4 0.15%
Seedling lethal 4 0.15%
Wide leaf 3 0.11%
Seed size 3 0.11%
Disease sensitive 2 0.08%
Single tiller 2 0.08%
Yellow green leaf 1 0.04%
Multiple tillers 1 0.04%
Coleoptiles shape 1 0.04%

49. MUTATION DETECTION WITH THREE CANDIDATE
GENES
 Screening of the targets of three candidate genes of Ppd-D1,
Rubisco activase A and Rubisco activase B in 512 random
plants from the M2 TILLING population obtained 15, 7, and
9 mutations, respectively.
 Sequencing confirmed that all of the mutations were G to A
or C to T transitions as expected from alkylation by EMS.

51. CASE STUDY 3
COTIP: Cotton TILLING Platform, a
Resource for Plant Improvement and
Reverse Genetic Studies
 Usman Aslam, Hafiza M. N. Cheema, Sheraz Ahmad, Iqrar A.
Khan, Waqas Malik and Asif A. Khan
 1 Plant Genetic Resources Lab, Department of Plant Breeding
and Genetics, University of Agriculture, Faisalabad, Pakistan,
 2 Genomics Lab, Department of Plant Breeding and Genetics,
Bahauddin Zakariya University, Multan, Pakistan

52. MATERIALS AND METHODS
Plant Material and EMS Treatment :
 For EMS treatment, 1,00,000 cotton seeds were de-linted
with conc. H2SO4 for each of PB-899 and PB-900.
 The percent solution of EMS @ 0.2% and 0.3% (v/v) was
prepared in ddH2O.
 The de-linted seed completely dipped in aqueous solutions
of EMS was kept at room temperature with continuous
shaking of 50 RPM for 3 hr.
 The mutagenized seed was washed twice with tap water, air
dried and grown in the field conditions.

53. ESTABLISHMENT SCHEME OF EMS INDUCED
COTTON MUTANT LIBRARY
 1,00,000 cotton seeds were EMS mutagenized that produces
overall on average 20,000 M1 plant progenies.
 One boll per plant selection was made from fertile plants. For
each M1 plant, 4–5 plants were raised as a M2 progeny. A
total of 5000 M2 progenies were raised.
 Only those plants from each progeny were tested for
TILLING, which produced viable bolls.
 DNA was extracted from plants of each of the progeny
labeled.
 Phenotyping was done on different plant observations.

54. DNA EXTRACTION, NORMALIZATION & POOLING SETUP
 The young leaf tissue samples of 8000 M2 plants were
collected at squaring stage and DNA was extracted individually
using modified CTAB method.
 The DNA samples were then electrophoresed on 1% agarose
gel.
 Mutational Frequency =
No. of samples screened Genomic region studied (kb)
No. of mutations identified

55. RESULTS AND DISCUSSION

56. OCCURRENCE OF INDUCED PHENOTYPIC VARIATION IN
IMPORTANT TRAITS OF THE MUTANT POPULATIONS.
Trait Variation No. of M2 plants
Branching pattern Spiral shaped
Bowl shaped
4
15
Plant stature Bunchy top
Umbrella shaped
Dwarf
Tall
3
7
157
8
Boll Type and shape Oval tiny
Green big
Purple color
33
6
1
Leaf shape Large palm
Fuse lobed shaped
Variegated
Irregular lobe symmetry
8
113
3
7
Fruit type Un-opened mature boll
Tri-loculed
Opened infertile bolls
340
730
870

57. Trait Variation No. of M2 plants
Anther Inflated hollow
Infertile
22
56
CLCuD tolerance Partial resistant
Resistant
Susceptible
230
86
150

58. MUTATION FREQUENCY ESTIMATION IN EMS MUTAGENIZED COTTON
POPULATIONS OF VAR. “PB-899” AND “PB-900.”
Sr
N.
Target tilled
gene
Gene
locus
no.
Genom
e size
per
gene
(bp)
No.of
M2
prog.
screene
d
No.of
mut.
identifi
ed (PB-
899)
Mut.
freq.
(PB-
899)
No.of
mut.
identi
fied
(PB-
900)
Mut.
freq.
(PB-
900)
1 Actin (GhAct) AF059
484
968 8,000 43 1/180
kb
30 1/258
kb
2 Sucrose
synthase
(GhSuS)
FB742
816
780 8,000 33 1/189
kb
18 1/346
kb
3 Pectin methyl
esterase
(GhPME)
JX003
001.1
995 8,000 3 1/2.65
mb
2 1/3.9
mb
4 Resistance
gene analogs
(GhRGAs
AY627
695
1000 8,000 4 1/2.0
mb
0 --

59. CONCLUSION
 The need for crop improvement to combat with biotic and
abiotic problems in agriculture is increasing with the
changing environmental perspectives.
 TILLING is a useful reverse genetic tool, used to create and
identify mutations in various organisms especially in plants
and successfully manipulated in several crop species.
 Furthermore, as EMS generates an allelic series of targeted
genes, it is possible to explore the role of desired genes that
are otherwise, not likely to be recovered in genetic screens,
based on insertional mutagenesis/transformation.

60.  TILLING and EcoTILLING are high-throughput and low-
cost methods for the discovery of induced mutations and
natural polymorphisms.
 The methods are general and have successfully been applied
to many plants, including crops.
 With sequence data and general tools such as TILLING,
reverse genetics can be applied to lesser studied species.
 Now that successes have been reported in a variety of
important plant species, the next challenge will be to use the
technology to develop improved crop varieties.