Authors: Manoj Kumar. Nalla*, Kranthi Tirlangi**, Rakesh Sharma V*, Gururaj B. Matapathi* and Pradeep K. Jatav*.
*Division of Vegetable science, ICAR- Indian Agriculture Research Institute, New Delhi-12
** Division of Fruit science, Dr.YSR Horticulture University, V.R. gudem-534101
Corresponding author: manojhorti@gmail.com

A genetic mutation is a very powerful tool that institutes a direct link between the biochemical function of a gene product and its role in vivo. By mutagenesis, recognition of genes and the role of their products can be determined by isolating and examining mutants that are defective in specific process pathway. The consequence of EMS mutagenesis often results in a large number of recessive mutations across the genome. A specific advantage of EMS mutagenesis is that a series of allelic mutations can be obtained, exhibiting a range of phenotypes that can serve as the basis of detailed structure function studies. The mutations by EMS mutagenesis are generated exposing genome-wide and can allow for a high degree of mutational saturation without the excessive collateral DNA damage. By furnishing a wide range of mutant alleles makes TILLING ( Target induced local lesions in genome)method potentially relevant to any organism that can be mutagenized. It can also be used in species which transgenic methods are limited or not applicable.

Procedure of TILLING method:

Basic TILLING method allows for high-throughput identification of single-base-pair (bp) allelic variations. The basic step begins with mutagenized seeds which are obtained from treatment with EMS. The ensuing M1 plants are self fertilized and the M2 generation of individuals is used to prepare DNA samples for mutational screening. The DNA samples are pooled, arrayed on microtiter plates and were subjected to PCR. The amplified products are incubated with an endonuclease such as CELI, a member of the S1 nuclease family of single strand-specific nucleases. CELI cleaves the 3’ side of mismatched DNA where the heteroduplex between the wild-type and the mutant strands of DNA loops out; homoduplexes are left intact. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed by examining the gel readout. Differential doubleend marking of amplification products allows for rapid visual ratification because mutations are observed on complementary strands and can be easily discerned from amplification artifacts. After detection of a mutation in a pool, the individual DNA samples are similarly screened to identify the plant carrying the mutation.

CELI, a plant-specific extracellular glycoprotein, has been shown to be suitable for genotyping applications because it preferentially cleaves mismatches of all types and has been used to detect heterozygous polymorphisms in DNA pools. Screening is performed on DNAs that have been arrayed in 96-well microtiter plates and pooled eightfold to maximize screening efficiency. Gene specific, fluorescently tagged primers are used to amplify pooled DNA. The amplification products are denatured and allowed to reanneal, generally by heating and cooling. As a result, a mutant strand will often reanneal with a wild-type strand, creating heteroduplexes at the site of the mutation or polymorphism. The resultant double-stranded products are digested with CELI, which cleaves one of the two strands at the heteroduplex mismatches. The cleaved products, are detected on polyacrylamide denaturing gels, identify individuals that have a mutation in the gene of interest. The size of the fragments carrying the 5’ and 3’ fluorescent tags can be used to estimate the position of the mutation within the amplicon.

Applications of TILLING:

TILLING, a very cheap and quick natural polymorphism breakthrough and genotyping method has advantages for determining the spectrum of variation and for genetic mapping based on linkage association analysis. In TILLING technique, if a mutation is detected in a pool, the individual DNA samples that went into the pool can be individually analyzed to identify the individual that carries the mutation. Once this individual has been identified, its phenotype can be determined. This technique works with good results even if a population contains pre-existing mutations that would compromise SNP discovery by other methodologies. This new screening method can be applied to several plant species whether small or large, diploid or allohexaploid and may provide a rapid approach to reverse genetics by identification of induced and naturally occurring variation in many species. TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants. These properties make TILLING approach a valuable tool for mutation analysis.

About Author / Additional Info:
My self pursuing Ph.D in vegetablescience in Indian agricultural research Institute.