Introduction to Random and

Insertional Mutagenesis

Dr. Amitha Mithra

Scientist
NRCPB
 Induced Mutations

For the first time HJ Muller demonstrated

that mutations can be induced. He used X-
rays to induce mutations. This is a very
significant step in Genetics which gave rise
to possibility of creating variation by man.

HJ Muller, Noble prize for

Medicine or Physiology, 1946

The agents that induced mutations are

called mutagens. The process on inducing
Muller with
mutations is called mutagenesis. This
his machine
gave rise to new phenotypes which for
subsequently helped in decoding the producing X-
inheritance of many genes. rays
Random Vs. Insertional mutagenesis for Functional Genomics

Item Random Mutagenesis (EMS) Insertional Mutagenesis

Mutant Resource Easy Difficult (highly technical)

generation
Raising mutant No restrictions Since transgenic in nature, have
lines to be raised in containment.
Large scale Possible Possible but field restrictions
screening for a apply
target trait
Identification of Difficult; have to go for map based Easy since sequence of the insert
mutated gene cloning; however NGS flanking sequence can be isolated
sequence technologies have given faster and
feasible approaches
Application of Possible Not possible
TILLING (a
reverse genetic
approach)
Genome Possible (one mutation/100 kb) Difficult owing to insertional bias
saturation
•EMS is a powerful mutagen – causes low mortality but high freq. of
mutations
•High density of nonbias irreversible mutations – saturation is possible
with relatively fewer plants
•Both loss and gain of function mutations
•A series of alleles and a series of degree of functionality
 TILLiNG - (Targeted Induced Local Lesions in Genome)
A reverse genetic approach for detecting point mutations

• TILLING was developed by McCullum et al., in 2000.

• It is a reverse genetic approach.
• Used for detecting induced mutations in genome in any species
• EMS is the most used chemical mutagen for developing a tilling population – can
target all genes – even in gene families and multi copy genes
• Homozygous backgrounds are preferred for detection; heterozygotes can also be
used .
• Detection system should be a sensitive one like DHPLC or LicoR – principle of
detection is same in both – both detect mismatches of heteroduplexes created by
melting and reannealing of heteroallelic DNA – missense and nonsense changes
are analyzed further.
• A judicious target selection for TILLiING is possible based on the gene sequence,
target of mutagen and changes it would cause – this would increase the
probability of identifying deleterious alleles.
• Conserved sequences and spice junction sites are also other preferred targets.
• Tool – coddle – codons to optimize discovery of deleterious lesions
Target selection for TILLING
 Basic steps involved in TILLING

1. EMS mutagenesis and raising M2

2. Preparing DNA stocks and pooling of individuals
3. Choosing candidate gens and their targets for PCR amplification
4. PCR amplification of the target region
5. Denaturation and reannealing to allow formation of heteroduplexes
6. Detection of SNP by DHPLC (a peak in the chromatogram) or LiCOR
7. Identification of mutant individual from within the pool
8. Sequencing and confirmation of the PCR amplicon
9. Phenotyping of the mutant
High Throughput Tilling using Cel1
enzyme and Li-COR
1. For detection DHPLC, or CEL1
based detection system (Li-
COR) can be used.
2. Primers are designed for the
gene of interest – longer primer
and higher Tm desirable.
(Target regions in the gene can
be predicted by a web based
tool called CODDLE).
3. Samples are pooled and a two
step mutation detection
process is carried out.
4. Denaturing and reannealing of
the PCR product is carried out.
This forms heteroduplexes
which are identified as extra
peak or band by DHPLC or
PAGE.
5. This has to be sequenced to
identify the mutation after
identifying the mutant
individual within the pool.
•For Li-COR detection flourescently labelled (infrared) primers are used and the
two primers are labeled with two different dyes.
•The heteroduplexes formed with the PCR products are treated with CEL1
enzyme which cleaves them at the point of mismatch.
•Samples are run in Li-COR till the sample completely runs off the gel.
•The fluorescent images are detected and the real time images are recorded by
the system.
•Since each fragment is labeled with the 2 different dyes, if there is a
mismatch and the DNA is cut, two smaller fragments will be present, one
labelled green, one red along with the wild type bands.
•These 2 smaller fragments will add up to the same molecular weight as the
wild type fragment. This is a check point to distinguish true mutants from
artifacts.
• Thus through this methodology, an almost exact identification of the
base pair where the mutation occurred is possible.
MutMap – A rapid NGS based method for gene
identification in EMS mutants
1. Generation of mutants (in crops with reference genome)
2. Crossing the mutant generated (M3 to M5) to the wild-type plant of the
same cultivar used for the mutagenesis.
3. The resulting F1 is self-pollinated to obtain F2 or more advanced
homozygous progeny segregating for the mutant and wild-type
phenotypes (most mutations are recessive and hence homozygosity
shouldn’t be an issue).
4. Phenotyping F2 - Crossing of the mutant to the wild-type parental line
ensures detection of phenotypic differences at the F2 generation between
the mutant and wild type – better to work out inheritance before embarking
on MutMap.
5. Principle: All the SNPs and InDels created in the mutant would segregate
in 1:1 ratio in the F2 population. This will be also the case in bulk of mutant
types. However the causal SNP alone would not segregate in the mutant
bulk.
6. DNA of F2 displaying the mutant phenotype are bulked (20 plants)
and subjected to whole-genome sequencing (preferably Illumina with >
10X coverage) followed by alignment to the reference sequence of the
wild type.
7. For success, substantial genomic coverage is important. 50% reads
shall be of wild type and 50% of mutant type in unlinked loci. This %
would tilt towards mutant type in linked loci and would be completely of
mutant type in the causal loci (total SNP changes would be in the
range of 950-2200 for rice in a mutant).
8. Thus the SNP index of the causal locus is 1 and nearly 1 in the
closely linked loci where SNP index is defined as the ratio between
the number of reads of mutant SNP to the total number of reads
corresponding to an SNP. Hence only regions with SBP index around
1 are calculated. Thus comparing SNP index of all SNPs against the
genomic regions reveals the causal mutant.
Identification of genomic regions harboring causal mutations for two pale green leaf mutants,
Hit1917-pl1 and Hit0813-pl2, using MutMap. (a) Leaf color and SPAD (stability of soil plant
analytical development) values (an estimate of chlorophyll content) of wild-type (WT)
Hitomebore and two mutants. Error bars, s.d. (b) SNP index plots for two leaf color mutants
(Hit1917-pl1 and Hit0813-pl2) showing chromosomes 10 and 1, respectively. Red regression
lines were obtained by averaging SNP indices from a moving window of five consecutive SNPs
and shifting the window one SNP at a time. The x-axis value of each averaged SNP index was
set at a midpoint between the first and fifth SNP.
MutMAP is more advantageous than the related technique – SHORE map

Item MutMap SHORE Map

crossing M x WT M X distant genotype (col M x
Ler)
SNPs in reads Looks for SNPs incorporated Looks for Col type SNPs
by EMS
Number of SNPs to Fewer (~ 1000-2000 in rice) More (~ 3 lakhs in Arabidopsis)
be analyzed
Population size 200 are sufficient to get 20 > 500 required to get 50 plants
mutant types of mutant type.

Traits All traits - even quantitative Only discrete traits could be

minor effect phenotypes can mapped because in a distant
be clearly distinguished from cross only qualitative traits can
the WT be easily discerned.
Reliability Subjected to sufficient More because clear cluster of
coverage SNPs with SNBP index of 1
could be identified
 MutMap+ for sterile and lethal mutations

• Those mutants that do not survive till the adult plant are identified. The heterozygous
individuals showing WT phenotype are chosen and their progenies are used for Whole
genome sequencing.

• M1 is heterozygote. Around, say 10 M2 lines are grown in the field and M3 lines harvested
from them. Since only the surviving adult plants can give rise to M3 this M2 is heterozygous
at mutant locus. The M3 ones are carefully phenotyped to identify WT and mutant and the
samples for DNA extraction are collected when the plants are young. Once the mutant type
and WT are identified (20-40 plants) in M3 from segregating M2s, they are bulked separately.
The M3 progenies for bulking should be from only one M2 individual. Both the bulks are
subjected to sequencing separately.

• The sequence of bulks and separately aligned with the WT sequence. SNP index vs
genomic regions (chromosome) graphs are generated.

• However SNP index of 1 is possible from two sources in mutant bulks – one, the causal
SNPS and the other, random fixation of SNPs to homozygosity (50%) – however the latter
ones would be homozygous in the WT bulk also.

• D of SNP index between the bulks is also calculated. This is nearly 0 for all the unrelated
homozygous regions and significantly >0 for causal SNP.
 Mutant bulk WT bulk

(A) Seeds harvested following EMS mutagenesis of rice at immature embryo stage are used to establish M1 generation,
at which stage most of mutations incorporated by EMS are in the heterozygous state. (B) M2 progeny obtained from a
self-fertilized M1 plant segregate for wild-type (indicated by green color) and mutant (brown color) phenotypes. Here we
focus on wild-type heterozygous individuals. (C) Heterozygous M2 plant are selfed to obtain M3 progeny that segregate
3:1 for wild-type and mutant phenotypes. Genomic DNA from 20–40 M3 mutant and wild-type M3 progeny are separately
bulked, and subjected to whole-genome sequencing. The resulting short reads are aligned to reference sequence of the
cultivar used for mutagenesis. (D) SNP-index is calculated for each SNP, and plots relating SNP index and chromosome
positions are obtained for both the mutant and wild-type M3 bulks separately. The two SNP-index plots are compared to
identify the region with SNP-index = 1 that is specific to the mutant bulk. (E) We can also evaluate D (SNP-index) plot,
which is obtained by subtracting SNP-index value of wild-type bulk from that of mutant bulk. Genomic region harboring
the causal mutation should have positive D(SNP-index) values.
 Insertional Mutagenesis

Principle: A DNA fragment with a known sequence is allowed to

insert into the genome (when it lands in a gene, it usually
causes a recessive, loss of function mutation).
T-DNA IM and
Transposon IM