High throughput TILLING for

Functional genomics

Anilkumar, C.PALB 5062

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

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 silent5% of mutations are truncations45% of mutations are mis-sense

• Overall, 10% of mutations cause a phenotypic change

• 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

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.

• Detection of MutationsDHPLC

• 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.

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

Breeding Science 61: 462–467 (2012)

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)

International Journal of Plant Genomics, 2011

Case study…

MethodsMutagenesis: Sorghum inbred line BTx623 was used

to generate the mutant populations using EMS (0.1 to 0.6% (v/v) mutagenesis.

• 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.

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

• 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

Thank you...