" TILLING : A Reverse genetic approach for crop improvement"

GENETIC APPROACHES FOR FUNCTIONAL GENOMICS

•         The main aim of genetic approaches to link between genotype and phenotype

•       There are mainly two approaches i.e., Forward genetics and Reverse genetics. Both these processes aim to determine the function of a gene

Forward genetics approaches: From phenotype to genotype

Forward genetics , in which one starts with a particular identified phenotype and the gene sequence is ultimately deduced through screening large numbers of individuals for phenotypic variations.

Reverse genetic approach : From genotype to phenotype

•  The gene sequence is known and mutants are screened to identify individuals with structural alterations in the gene of interest.

•   Reverse genetic approach is generally less time demanding Reverse genetic strategies have been successfully used for functional genomics in many animal and plant species. The widespread availability of sequence data allows researchers to rapidly design reverse genetic strategies to determine gene function.

Methods to reverse genetic approaches:

•         Homologous recombination

•         Agro-bacterium mediated insertional mutagenesis

•         Transposon tagging

•          Rnai (RNA interference) or PTGS (Post Transcriptional Gene Silencing)

•         Chemical mutagenesis followed by detection of mutation through TILLING

•         Detection of natural polymorphism by EcoTILLING.

TILLING (Targeting Induced Local Lesions IN Genomes) is a non-transgenic reverse genetic technique that is suitable for most plants. For TILLING, mutations are created by treatment with the same chemical mutagens that have been successfully employed in mutation breeding programs for decades. By using chemical mutagens that induce primarily random point mutations at high density, allelic series of missense and truncation mutations can be discovered with TILLING. Thus with only a small population, multiple alleles may be obtained regardless of the size of the gene. Gene regions are targeted for mutation discovery using PCR and standard SNP discovery methods. The use of general techniques for the generation and discovery of mutations means that the method should be applicable to a wide variety of organisms. TILLING methodology can also be used to uncover natural nucleotide variation linked to important phenotypic traits, a process termed EcoTILLING. The current status of various plant TILLING and EcoTILLING projects discussed in this review show that the methods are generally applicable across the plant kingdom.

Steps in TILLING:

1) Development of a mutagenized population

 2) DNA Extraction and pooling

 3) Mutation discover

Developing a Mutagenized population:

•         Plants are ideally suited for TILLING

•  Traditional mutagenesis techniques have been used to create many new crop varieties [ Stadler, 1928]

•         The ideal mutagen is one that randomly induces single nucleotide substitutions, or INDELs at a high frequency in genome

Chemical mutagenesis:

Among the mutagens, chemical agents play a major  part and have become popular.

Ø Ethyl methane sulfonate (EMS)

Ø  Methyl methane sulfonate (MMS) 

Ø  Ethyl nitroso urea (ENU)

Ø  Sodium azide

Ø  Methyl-Nitrosourea ( MNU)

Alkylating agents (EMS), which yield  predominantly point mutations, have been especially  valuable for TILLING.

Selecting Tissue for Mutations:

•         The optimal tissue is one that produces non-chimeric plants harbouring a high density of induced mutations

•         Seeds are frequently used

•         But in maize pollen will be treated with the mutagens

Mutagenized population:

•         Mutagenized seeds are sown and that generation is M1 which is highly chimeric in nature

•         Selfing of individual M1 plants to get non-chimeric M2 Population

•         M2 is a ideal Mutagenized population

•         When pollens are used M1 population is not chimeric and can directly used for DNA extraction.

DNA extraction:

•         Genomic DNA samples are typically extracted from individual M2 plants

•         The DNA extraction method used for TILLING should produce a sufficient yield and quality of genomic DNA 

DNA Pooling:

•         Samples are normalized to a common concentration

•         Then pooled together to reduce the cost of mutation screening (1-8 folds)

•         Pooling of up to eight samples has been used for TILLING

•         Pooling of samples is advantageous for  these reasons:

          (1) More potential heteroduplexes can be seen

          (2) The number of individuals that can be surveyed at a time is increased

Mutation Discovery:

•         Generally Mutation discovery is nothing but SNP Discovery (Mismatch Discovery)

SNP discovery technologies include

•         Array-based methods

•         Denaturing HPLC

•         Mass spectroscopy

•         Denaturing gradient capillary electrophoresis and

•         Enzymatic Mismatch Cleavage

SNP discovery through enzymatic mismatch cleavage  :

•         The most common method used is enzymatic mismatch cleavage  followed by resolution on PAGE to detect the cleaved fragments

•         The cleaved fragments are detected by  fluoresce The pooled DNA is arrayed into 96 well microtiter plates

•         Pooled samples are amplified using primers targeting the gene of interest

•         The forward and reverse primers are differentially 5’ end labelled with IRD700 and IRD800 dye labels

•          Dyes are fluorescent labels which can be detected  at ~700 nm and ~800 nm, respectively.

•         Next, heteroduplexes and homoduplexes are formed from the PCR products of pooled samples

•         The endonuclease enzyme CEL I is applied and a short incubation is required for the enzymatic reaction 

•          CEL I not only specifically recognizes mismatches in the heteroduplex, but it also cleaves DNA on the 3’ side of the mismatch

•         Therefore, when fragments are separated a full length product (detected in both 700 and 800 channels)  Two cleaved fragments (one IRD700 labelled, one IRD800 labelled) will be visible

•         The sum of the cleaved fragments should equal the full length PCR product

•         The size of the cleaved fragments can be estimated by comparison to a size standard

•          The approximate location of the mutation will be identified and further confirmed by sequencing The CEL I nuclease extracted from celery is most commonly used  endonuclease (Oleykowski et al., 1998)

•         CEL I is a single-strand specific nuclease related to S1 nuclease

•         CEL I, S1 and Mung Bean nucleases have all been shown to be usable for mutation discovery (Till et al., 2004).

•         Detection of digested fragments obtained by separation on a denaturing PAGE attached to a LI-COR 4300 DNA analysis system

•         Pools containing an induced mutation will consist of a mixture of homo and heteroduplexes

Merits:

´ Cheap and rapid reverse genetic approach

´ Detection of mutation at single nucleotide level

´ Independent of genome size and used for most of the organisms.

Demerits:

´ Skilled labour are required

´ Low rate of induction of mutation

 

Conclusion:

TILLING and EcoTILLING are high-throughput and low-cost methods. The methods are general and have successfully been applied to many plants, including crops. With sequence data and general tools such as TILLING, reverse genetics can be applied to lesser studied species. Now that successes have been reported in a variety of important plant species, the next challenge will be to use the technology to develop improved crop varieties.The utility of induced  mutations and  natural polymorphism has already been established for crop breeding and so the  task is mostly one of implementation.