Allele mining ,Tilling and Eco Tilling

Presented by:-

Jadhao Kundansingh R.

09ABT/11

Department of Agril-biotechnology,Orissa University of Agriculture & Technology,BBSR.

WHY ALLELE MINING

HOW DIFFERNTIATE GENE FROM ANOTHER?

WHAT IS THE ALLELE MINING?

Identification and access to allelic variation that affects the plant phenotype is of the utmost importance for the utilization of genetic resources, such as in plant variety development.

Considering the huge numbers of accessions that are held collectively by gene banks, genetic resources collections are deemed to harbour a wealth of undisclosed allelic variants.

The challenge is how to unlock this variation. Allele mining is a research field aimed at identifying allelic variation of relevant traits within genetic resources collections.

IMPORTANCE

It helps in tracing the evolution of alleles Identification of new haplotypes and

development of allele-specific markers for use in marker-assisted selection

This capability will be important for giving rice breeders direct access to key alleles conferring

(1) resistance to biotic stresses, (2) tolerance of abiotic stresses, (3) greater nutrient use efficiency, (4) enhanced yield, and (5) improved quality, including human nutrition

It can also provide insight into molecular basis of novel trait variations and identify the nucleotide sequence changes associated with superior alleles. In addition, the rate of evolution of alleles

Breeding Synopsis

STEPS IN ALLELE MINING

primer designing

TILLING METHOD

The TILLING Method. Seeds are treated with a chemical mutagen to induce genetic variation, and then planted. Theresulting M1 population of plants is chimeric for mutations. Therefore, one seed from each M1 is planted to create the M2 population.

M2 DNA is extracted from leaf tissue DNA samples are pooled to increase throughput and PCR amplified with dye-labeled PCR primers specific to a target gene of interest. PCR products are denatured and allowed to reanneal to form heteroduplexes. Heteroduplex DNA is then cleaved by Cel I and analyzed

Illustration of a Cel I cleavage reaction. PCR primers that have been end-labeled withtwo different color dyes (red and green arrows) are used to amplify a targeted region of the genome in a pool of DNA consisting of multiple individuals. After PCR, DNA fragments are denatured and allowed to reanneal to form homoduplexes and heteroduplexes. Cel I is added to the reaction and cleaves DNA 3’ of the mismatch. The cleavage reaction is concentrated, denatured and separated electrophoretically on a LI-COR DNA analyzer.

ECO-TILLING

DNA from many (eight) plants are pooled, The amplified products are denaturated byheating and cooling slowly for randomly re-annealing and forming homo- and heteroduplexes, double-stranded products are digested by CELI endonuclease, ---Gel Electrophoresis

APPLICATION

Thank you