studying gene function by induced mutants

1. Anilkumar, C.
PALB 5062

2. One of the most important breakthroughs in the
history of genetics-mutations can be induced (Muller,
1927; Stadler, 1932).
Identification of genes and the function of their
products can be determined by isolating and studying
mutants.
TILLING (Targeting Induced Local Lesions In Genomes)
combines chemical mutagenesis (Koornneef et al., 1982)
with a sensitive mutation detection instrument.
Concept of TILLING was introduced in 2000, using the
model plant Arabidopsis thaliana (Mc Callum et.al.,
2000).
It provides a non-transgenic method for reverse
genetics.
Introduction

3. How TILLING Works…?
 Basic TILLING method allows for high-throughput
identification of single-base-pair (bp) allelic variations/
point mutations.
Steps:
Mutagenesis- Most important step.
• Among the mutagens, chemical agents play a major
part and have become popular.
• Alkylating agents (esp. EMS), which yield
predominantly point mutations, have been especially
valuable for TILLING.
50% of mutations are silent
5% of mutations are truncations
45% of mutations are mis-sense
• Overall, 10% of mutations cause a phenotypic change

4. • The resulting M1 plants are self fertilized.
• The M2 generation of individuals is used to prepare
DNA samples for mutational screening.
• Pooling of Samples
• in order to check many samples for a possible
mutation, samples must be pooled

5. After pooling, PCR begins...
• PCR
• Primers must be carefully selected to ensure
that they are going to amplify a suitable
region
» don’t want to amplify non-coding region
» use of a longer primer and high Tm
helps to increase specificity.
• End step of PCR is to denature all DNA
present, then reanneal
» this causes a small bubble to form
between mismatched pairs of DNA
(where the mutation has occurred)
forming a heteroduplex.

6. • Detection of Mutations
DHPLC
• This is the method used originally.
• Can detect hetero duplexes with good efficiency.
• Not as useful for high throughput because of the
time required to run a sample.

7. Enzyme Cel-1 :
• Derived from celery
• Cuts DNA at a mismatch
(hetero duplex); cuts at 3’ end
of mismatch
• Cel-1 is added to the final PCR
products and cuts at bubbles
formed in hetero duplexes
ATGCGGACTG
|||||| |||
TACGCCGGAC
ATGCGG CTG
|||||| |||
TACGCC GAC
Cel 1+
© 2005 Prentice Hall Inc. / A Pearson
Education Company / Upper Saddle River,
New Jersey 07458

8. Breeding Science 61: 462–467 (2012)

9. Mutant Discovery in TILLING Populations
• Direct Sequencing
• Li-Cor
• High-Performance Liquid Chromatography
(HPLC)
• Electrophoresis
• High-Resolution Melt (HRM)
• MALDI-TOF
• Next-generation sequencing(NGS)

11. International Journal of Plant Genomics, 2011

12. Case study…

13. Methods
Mutagenesis: Sorghum inbred line BTx623 was used to
generate the mutant populations using EMS (0.1 to 0.6%
(v/v) mutagenesis.

14. • A total of 768 mutant lines were assayed for mutation induction in
the target genes
• Four gene targets were selected based on their potential
contribution to bioenergy, nutrition, and agronomic performance for
high throughput TILLING (table 2).
• Eight-fold pools of genomic DNA from leaf tissues of M2 plants
were used for TILLING.
• PCR, TILLING, and fragment separation(Li-COR gel system)
• Histochemical analysis of cell wall.

15. Results:
• 0.25% EMS concentration is most effective.
• A total of five mutations were detected by TILLING in four gene
targets
No mutations were detected for one of the targets, 1-
aminocyclopropane-1-carboxylate oxidase (ACO1).
All five mutations detected by TILLING were re-examined by
morphological observation and re-sequenced to verify the results

16. • The mutations revealed via TILLING from the gene targets
COMT and PHYA were all determined to be missense
mutations and found to be present at different positions in
exon 2.
• This induced mutation altered the codon from a hydrophobic
glycine amino acid to a hydrophilic serine amino acid , which
may affect the protein structure.
• Alterations in the COMT gene are associated with bmr
mutations in sorghum, characterized by a brown midrib with
reduced lignin content and increased digestibility