This document discusses TILLING and Eco-TILLING techniques for crop improvement. It describes the limitations of existing gene knockout methods and outlines the TILLING workflow, including DNA pooling schemes and both conventional LI-COR gel-based and newer Illumina sequencing-based methods for detecting mutations. Eco-TILLING can be used to mine germplasm for SNPs and assess heterozygosity to uncover variants related to disease resistance or gene function. Both TILLING and Eco-TILLING can help characterize natural variation and are useful techniques in plant breeding programs.

1. TILLING AND ECO-TILLING
FOR CROP IMPROVEMENT

6. WHY TILLING…….?
 The present methods for targeted gene inactivation are
RNAi and T-DNA insertion.
 Transgenic RNAi technology or introduced transposons
complicate field testing and may not be accepted by the
consumer.
 To overcome the limitations of knocking out the entire
gene and to expand knowledge of active gene mutations.
 Regenerating whole plants through tissue culture is a
practical hindrance to apply these technologies.

9. TILLING Workflow:

10. DNA Pooling scheme
One dimensional pooling –
Each plant is represented in a single pool of eight
768 unique samples (96 pools) can be screened in a single 96 well
TILLING assay.

11. Two dimensional pooling
 samples are arrayed in 12 x 8 grids
 Pooling by combining samples by columns and rows
 An individual sample is represented in two unique pools in
the assay

15. Conventional LI-COR (Gel) based
TILLING

16.  Samples are loaded onto a comb using either a robot or
manually with a pipette onto the LI-Cor gel.
 LI-COR gel running machine detects fluorescent tags
on fragments and creates a real time image of the gel as
it runs
 Since each fragment is labeled with the 2 different dyes
and if there is a mismatch and the DNA is cut, two
smaller fragments will be present, one labeled red, one
green (IRDye700 and IRDye800).

17.  Once the mutation is detected then based on the pools, DNA will be
identified at individual plant level either by restriction analysis or Sanger’s
sequencing of gene product from eight individuals (pool of 8 columns in 1
well).
 Limitations of LI-COR gel analysis
This method is time consuming, expensive because fluorescent primers
are used for screening of mutation.
 To overcome the Li-Cor based analysis now the next generation
sequencing (Illumina Sequencing) and bioinformatics tools are in use to
detect the mutation in chosen gene or in the set of genes.
 Illumina Sequencing is less time consuming, less expensive because
fluorescent primer are not required.

18. ILLUMINA SEQUENCING

34. Advantages Eco-TILLING
 Valuable tool for mining for SNPs in germplasm,and in
assessing heterozygosity,
 Uncovering variants for disease resistance,
 Ascertaining the function of a gene or regulatory element by
detecting natural variants.
 EcoTILLING can also be a good technique to employ
especially when working with a well established population
with thoroughly characterized morphological data.

35. Utilization in breeding program