Allele Maining in Plant Breeding Technology

Allele mining is a process in plant genetics and breeding for discovering, identifying, and utilizing novel or favorable alleles from diverse germplasm, mutant libraries, or wild relatives to improve crop traits such as yield, disease resistance, and stress tolerance. This approach uses methods like genome-wide association studies (GWAS), SNP arrays, and gene sequencing to identify new genetic variants that enhance plant architecture, productivity, and adaptation, leading to the development of superior crop varieties with increased genetic diversity and improved agronomic performance.

1. Steps in Allele Mining:
a) Selection of target trait (Trait Priority)
b) Identification of accessions linked with desired phenotypic trait
c) Selection of genes underlying the chosen target trait (Gene Targets)
d) Primer designing for whole length of gene
e) PCR amplification from the identified accessions
f) Sequencing- based mining
g) PCR
h) Sequencing and identification of variation
i) Comparison of sequence data with phenotypic data and identification of superior alleles.
Allele mining done in three approaches / methods
1.Tilling
2.Ecotilling
3.Sequencing based
1)
2)TILLING APPROACH :

Procedure of TILLING Procedure of TILLING
Steps in Tilling :
1.Mutagenesis
2.DNA Preparation and pooling of individuals
3.PCR amplification of a region of interest
4.Denaturation and annealing – formation of Heteroduplex
5.Detection of Heteroduplex by endonuclease
6.Identification of the mutant individual
7.Sequencing of Mutant PCR product

2. ECO TILLING : a method for identifying natural genetic variations (polymorphisms) in a population,
particularly in plants. Unlike TILLING, which focuses on induced mutations, EcoTILLING aims to detect
naturally occurring mutations and variations. 
Requirements for EcoTILLING
-population (collection of natural cultivars/accessions/wild/elite accessions)
-DNA from the above population (mixed with a reference DNA to identify polymorphisms using the TILLING
process)
-Gene of interest, specific primers([email protected])
Steps in Ecotilling :
1.population Selection: A population with a diverse range of natural variation is selected. 
2.DNA Extraction: DNA is extracted from individual plants or tissues within the population. 
3.PCR Amplification: The target region of interest is amplified using PCR. 
4.Mutation Screening: Various techniques like single-strand conformation polymorphism (SSCP) or denaturing
gradient gel electrophoresis (DGGE) can be used to screen for mutations or polymorphisms. 
5.Sequencing: Sequencing is used to confirm the presence and location of mutations. 
Comparision of TILLING and ECOTILLING

3. 3) SEQUENCING APPROACH :
Sequencing-based allele mining involves PCR-based amplification of alleles of a gene in varied
genotypes and then DNA sequencing to recognize nucleotide variance in the alleles (Figure 4). Various alleles
among the cultivars through this approach can be identified. The method would help to analyze individuals
for haplotype structure and diversity to infer genetic association studies in plants. Applying this approach to
recognize the effect of mutations on gene structure, the sequences are analyzed for the location of point
mutations or SNPs and insertions or deletions (InDels) to build haplotypes.
Applications of allele mining: 
1 Detection of superior alleles. Characters vary in their degree of excellence and effectiveness in positive
or negative direction due to the allelic variation. allele mining helps us to asses a superior or most
performing allele for a trait, i.e., disease resistance, height, flood resistance.
3)Similarity analysis in inter and intra species. To find out similarities between species and within a
species allele mining can help us out. It incorporates us to arrange or classify the collection of
genomes.
4)Functional molecular marker development for MAS. Allele mining helps us to develop markers for
selection and hybridization purposes, as we know the sequences of DNA.
5)Identification of new haplotypes. The allele mining is a tool which evolves new variation which were
unknown to world before it gives us discovery to new haplotypes and allelomorphs.
6)Evolutionary study. Allele mining unfolds all the alleles present for a trait in nature, so it becomes
very easy to study the evolution of specific organism or plant.
7)Promoter mining is done for expression study and gene prediction. Allele mining helps us to predict
the gene behaviour and expression on the basis of currently available genome collection data and
the number of known alleles.