The document describes the TILLING (Targeting Induced Local Lesions IN Genomes) technique for functional genomics and bioinformatics in cotton. TILLING involves mutagenizing a population, typically using EMS to achieve one mutation per 300,000 base pairs. DNA is extracted from the mutagenized population and pooled for PCR amplification. The PCR products are then digested with CEL I enzyme to detect mutations based on mismatches. TILLING can generate an allelic series of mutations to provide insight into gene and protein function beyond what a single knockout could reveal. The technique is effective for crop improvement by creating beneficial changes without foreign DNA insertion.

1. (TILLING) FOR COTTON FUNCTIONAL GENOMICS
AND BIOINFORMATICS”
Aqsa Hafeez Khan.
Roll No:8798
MS Bioinformatics

2. THE AUTHORS
 Elizabeth A. Greene
 Currently working in bioinformatics field at the Fred Hutchinson
Cancer Research Centre in Seattle Washington.
 Luca Comai
 Provided space for growing plants used in experiments carried out
for this paper.

3. AIM OF THE PAPER
 To introduce a new technique useful for creating an allelic series of gene
disruption/knockout to the scientific community.
 Raising interest in the technique to generate new ideas for improvement
of TILLING, and expand TILLING to other organisms.
 Provide insight into possible uses for TILLING, such as genetic
modification of crops.

4. TILLING OVERVIEW
 Mutagenesis
 First you need to have a mutagenized population
from which to begin the process.
 Typically, you want to have a rate of one mutation
per 300,000 bp when creating your population.
 EMS is the mutagen used most often.

5. TILLING OVERVIEW
 Mutagenesis
 Most important step because if you don’t have a
good population to begin with, the rest of the
procedure is a waste
 50% of mutations are silent
 50% of mutations are not silent

6. General TILLING Technique
 The general procedure is as follows:
 A population is mutagenized with EMS (or another mutagen), and
allowed to grow up and produce progeny.
 DNA is isolated from the progeny of the mutagenized individuals.
 Then the DNA samples are pooled to reduce the number of PCR
reactions necessary for the detection of a mutation.
 These PCR products are subsequently digested with Celery CEL 1.

9. MECHANISM OF TILLING

10. TILLING OVERVIEW
Gel Image

11. TILLING WITH BIOINFORMATICS

13. “www.proweb.org”

28. high Peak shows ideal place for
mutations to caused
Moderate Peak shows average result
for mutations to caused
Low Peak shows bad place not
favorable for mutations.

35. RESULTS OF TILLING
 Allelic Series Created
 Due to different mutations causing either
truncations, single amino acid changes, etc, mutations
affecting the protein of interest are varied.
 this allows for an allelic series which may cause differing
phenotypes and allow for greater understanding of protein
function than a single knockout could provide.

36. FUTURE OF TILLING
 Detection of polymorphisms
 detection of mismatches can provide excellent
detection of polymorphisms due to the
mismatch of different alleles.
• Crop Improvement
• Allelic series can cause change in protein
function that could be beneficial
• Not having addition of foreign DNA alleviates
many worries for consumer groups

37. SUMMARY
• TILLING is an effective technique to use to
gain insight into gene function.
• TILLING is adaptable to a high throughput
environment.
• TILLING continues to evolve and improve
as a technique.

38. TILLING is
done!!

40. ANY QUESTIONS?