Allele miningDepartment of Agricultural Biotechnology,

OUAT, BBSR

Presented by,Arjun S. Pimple

07ABT/13

ALLELE?

Allele miningAllele mining

is a research field aimed at identifying allelic variation

of relevant traits within genetic resources collections

Some Facts• In human beings, 99.9 percent bases are same.• Remaining 0.1 percent makes a person unique.

– Different attributes / characteristics / traits • how a person looks, • diseases he or she develops.

• These variations can be:– Harmless (change in phenotype)– Harmful (diabetes, cancer, heart disease, Huntington's disease, and

hemophilia )– Latent (variations found in coding and regulatory regions, are not

harmful on their own, and the change in each gene only becomes apparent under certain conditions e.g. susceptibility to lung cancer)

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.

IMPORTANCEIt helps in tracing the evolution of allelesIdentification 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

Continue…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.

STEPS IN ALLELE MINING

Methods of Allele Priming

Eco TILLINGPCR Based Allele Priming

The TILLING Method.Seeds are treated with a chemical mutagen to

induce genetic variation, and then planted. The resulting 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

ECO-TILLINGDNA from many (eight) plants are pooled,

The amplified products are denaturated by heating and cooling slowly for randomly

Re-annealing and forming homo- and heteroduplexes, double-stranded products are digested by CEL-I endonuclease, Gel Electrophoresis

SNPs (A SNP is defined as a single base change in a DNA

sequence)

SNPs are found in coding and (mostly) noncoding regions.

Occur with a very high frequency-about 1 in 1000 bases to 1 in 100 to 300 bases.

The abundance of SNPs and the ease with which they can be measured make these genetic variations significant.

SNPs close to particular gene acts as a marker for that gene.

SNPs in coding regions may alter the protein structure made by that coding region.

SNPs may / may not alter protein structure

APPLICATION

WHY IT IS IMPORTANT?

Vaughan (1994) describes the genus Oryza and related grasses (subfamily Oryzoideae), while Kellogg (1999) summarizes the evolutionary relationships among the grasses in general, including the cereals.

The feasibility of PCR-based allele mining for stress tolerance genes in rice and related germplasm.

(CASE STUDY)

Nipponbare is a japonica cultivar and therefore belongs to isozyme group VI of O. sativa (Glaszmann 1987). For each gene, the Nipponbare allele will be most closely related to the alleles of other japonica cultivars and then progressively less closely related to alleles.

1) the isozyme groups I-V of O. sativa, (2) the other AA genome species, (3) the non-AA genome species of genus Oryza, (4) related grass genera such as Porteresia, and (5) the other cereals.

MATERIAL USED

The germplasm used in this study of allele mining comprised 64 accessions, including at least two representatives of each of the six isozyme groups of O. sativa, two accessions of each of the seven other AA genome wild species, 1 or 2 accessions of each of eleven non-AA genome wild species, one accession of each of five closely related grass genera, and 1-3 accessions of each of five other cereals.

Three geneThey evaluated their protocols for allele mining by

focusing on three genes important in abiotic stress tolerance. These genes encoded (1) calmodulin (Calmod, Z12828), (2) a late embryogenesis abundant protein 3 (LEA3, AF046884), and (3) SalT (Z25811).

Calmodulin is a part of the network of signal transduction pathways centered on calcium ions as second messenger in stress tolerance (Epstein 1998). LEA3 accumulates in cells to protect them against ionic changes accompanying various stresses (Skriver and Mundy 1991).

SalT accumulates in rice leaf sheaths and roots in response to salt and drought but its role is not clear (Claes et al. 1990). Both LEA3 and SalT are induced by ABA treatment (Moons eta!. 1995).

Sequences of PCR primers for amplification of three genes Serial Primer/Gene Sequence 5’-3’ Product size

1 Calmed 5’3’ CGC GCG CGC CFG CGT CGC CAA TOG 1254 bp

CGATGC TFC AAC TTA CTT GGC C

2 Clamod NC ATG GCG GAC CAG CTC ACC GAC GA 1178 bp

CAC CAT CAA CAT CGG CCT GAC CG

3 LEA3 5’3’ GCTTAG GAT CAATGG CTT CCC ACC 941 bp

CCAAAG GGAAAT CAT TCA CGG CGT C

4 LEA3 NC CTACCG CGC CGG CGA GAC CA 838 bp

TCC CTC GCC GTC GTC TCC GT

5 SalT 5’3’ CCA CGAAGACFATGA CGC TOG TG 574 bp

CTF TGA CCA CTG GGAATC AAG G

6 SalT NC ATGACGCTGGTGAAGATFGGCC 534bp

GOT GGA CGTAGATGC CAATTG C

Result