Abstract:
The Targeting Induced Local Lesions in Genomes (TILLING) technique is being put to use for elucidating gene functions to affect genetic improvement in cotton. The proposed research focuses on the generation of mutations in cotton (G. hirsutum) using chemical mutagen, determination of mutation in the targeted genes and the utilization of the useful mutations in the cotton improvement programs.
Problem statement
Cotton is prone to many insect pests and diseases and thus its production and quality has been constantly under threat. There is a need to effectively expand and spot the desirable genetic variability for the target traits.
Objectives of the Project:

1. Optimization of mutagenesis and development of a TILLING population
2. Identification of the mutation in pectin methyl esterase, actin, sucrose synthase, resistance gene analogues (RGAs) like NBS-RGAs and STK-RGAs and defense gene analogues (DGAs).
3. Utilization of the useful mutant for a targeted trait in the ongoing breeding programmes.

Methodology

• Development of kill curve, designing primers for targeted genes and preparation of CEL1 enzyme.
• Development of TILLING populations and carrying out pilot to estimate mutation density.

• Identification of mutants for the targeted genes in the TILLING populations.

Results and Discussions
For the construction of Kill Curve (LD50), a greenhouse experiment was conducted on three different species of cotton. Four genotypes from each of Gossypium hirsutum, barbadense and arboretum were treated with 4 doses of gamma rays and 4 concentrations of EMS.
Five doses of gamma rays and five concentrations of EMS were use for making the kill curve. The data was collected on germination percentage, plant height and different yield related traits and was analysed using nested design. The optimum dose for seed treatment was selected on the basis of at least 50% death of treated plants and production of viable seeds at that specific dose. According to these criteria, about 0.3% EMS dose was observed to be effective for PB-899 and 0.2% was observed to be effective for PB-900. 
In order to TILL the targeting proposed gene families [Actin, Sucrose Synthase, Pectin Methyl Esterase (PME), Resistance Gene Analogues (RGAs), and Defence Gene Analogues (DGAs)], DNA sequences along with CDS statements were screened from the publicly accessible databases available at NCBI website http://www.ncbi.nlm.nih.gov/.  The Intron-Exon models for respective DNA sequences of mentioned gene families have been determined and the primers designed at the functionally EMS affected region using the CODDLE software available at http://www.proweb.org/coddle/. The CEL1 enzyme needed for the DNA cleave at the mismatches to identify the mutations in the cotton TILLING populations was purified in the lab from the celery (Apium graveolens L.) using the standard protocol available at web page http://labs.fhcrc.org/moens/protocols/ Tilling%20Protocols/CEL1purification.pdf.
Conclusions:
Both gamma rays and EMS were effective in the induction of mutations in the genotypes of three cotton species. The EMS was comparatively more effective for the G. hirsutum species. More difference wesre found among species and even among genotypes within species in response EMS. The TILLING primers for target genes were tested and considered useful for the screening of mutant populations of upland cotton for point mutations. The CELI enzyme assay appeared useful for identification of single nucleotide mismatches and SNPs among species or within genotypes of species.
Recommendations:
The chemical mutagen EMS is effective in inducing useable variation at 0.2 – 0.3 % concentration