Higher plant genomes are very complex in nature. Most of them harbor polyploidy and large genomes. Deciphering these sequences require huge investments in terms of time and other resources. Once a plant genome sequence is available, it becomes imperative to study its gene content, number and function and its relationship with organisms both within and outside the species and genera. This part of deciphering the function of a gene is termed as functional genomics especially when the target gene will be used for developing a useful product in terms of adding value to the plant by improving its yield. Plant yields are generally ravaged by the combined effect of abiotic stresses like drought, salinity, high and low temperature, oxidative stress etc. Other important stresses include the biotic stresses including attack of insect, fungal, bacterial, nematodes etc. Often these losses lead to significant decline in the crop yield and in turn lead to shortage of food grains. Thus there is a constant pressure on the researchers to develop new products carrying genes from other sources and imparting the plant some advantage in terms of imparting tolerance to the biotic and abiotic stresses.
Assessing gene function requires painstaking work over a number of years. These require either targeting a particular gene for which some function is known or finding out new genes from a desirable genotype using mutations. Thus forward and reverse genetics are defined as below;
Forward genetics:Determining the gene function by following the phenotype to gene approach
• In forward genetics a set of mutants lines are created either by EMS and/or T-DNA /Transposon preferably called as knockout for the latter.
• These mutant lines are then screened for a particular phenotype using mutant screens or procedures where a large number of mutants can be screened in a bulk manner.
• The selected mutants are then carried forward and analyzed by PCR (in case of T-DNA insertion lines), backcrossed to remove or dilute any unwanted insertions of T-DNA.
• The sequence information generated from the PCR is then used to identify the gene where the insertion has taken place and this becomes the candidate gene for the phenotype tested in previous steps.
• The next logical steps involves cloning of the full length gene and cDNA(using RACE) from the wild type plants.
• In the final validation step, the gene isolated in the previous step is used to transform the mutant plant and phenotyped. If the mutant plant exhibits recovery of wild type phenotype, it is assumed that the wt type gene is able to rescue the mutant; hence this validates the gene function.
• In case of EMS induced mutants, the approach slightly differs: The mutants identified are used for developing mapping population.
• This mapping population is then genotyped and phenotyped and then the locus controlling the trait in question is fine mapped to a manageable window of less than 250 kb or so.
• Using various approaches then the entire region in this mapped interval is sequenced and aligned with wt to find out the mutant base.
• Validation then proceeds in a similar way as described for the T-DNA mutants.
Advantages and disadvantages of this approach:
• It is a tested method and many genes have been isolated in plants.
• It works very well in phenotypes that can be distinguished quickly, for eg. color, shape etc.
• It works in a unbiased manner because it is random in nature.
• The mutant lines created are quite stable and T-DNA is known to preferentially integrate in and around the genic region.
• On the other hand, phenotypes that are difficult to evaluate make it very tedious.
• Only one gene/trait can be analyzed.
• Some genes may be missed and the procedure works only in plants that are amenable to Agrobacterium transformation.
Reverse genetics: Determining the gene function by gene to phenotype approach. This strategy works on the single gene for which the function is to be determined.
• A gene sequence is selected for which the function or the phenotype it creates are not known
• Mutations are created in this gene only by targeting it generally at RNA level leading to suppression of the native mRNA produced by the gene.
• This results in a phenotype that can be easily assigned to the gene in question.
• RNAi (RNA interference) and TILLING (Targeting Induced Local Lesions IN Genomes) are some of the common techniques used in this approach.
• RNAi is characterized by designing constructs carrying the sequences of the target gene in an antisense orientation. This construct is mobilized either through viruses or agrobacterium. The RNA produced from this construct then binds to the target gene resulting in no translation, loss of function and a phenotype is created.
• TILLING relies on chemical mutagenesis agents such as EMS. In this technique EMS mutants are created and then individual mutants are pooled into groups. Then these mutant pools are analyzed for a particular gene by PCR amplification. The amplicons from the mutant pools are then hybridized to wt amplicons. Any change in the sequence leads to the formation of the heteroduplex which can be detected by various analytical techniques such as dHPLC or LICOR gels and then the mutant plants can be analyzed both for the phenotype and the location of the mutation.
Advantage and Disadvantages:
• It works wonderfully for known gene sequences in short span of time.
• Screening of large population of transformants is not required as it is targeted approach.
• Very useful in polyploid systems.
• On the other hand, silencing may not be total and some leakage may be observed.
• The stability of the RNAi based suppression may vary in the future generations and needs constant monitoring.
• Some other genes sharing homology with the target gene may also get knocked out leading to multiple phenotypes.
Thus reverse and forward genetics approaches working in tandem are very essential for determining gene function. Both the approaches have their own pros and cons but a number of agronomically important genes have been discovered in plants using these. Combined with the power of microarrays and next generation sequencing platforms, these approaches have acquired newer dimensions and utility in the area of functional genomics.
1. Reverse genetics techniques: engineering loss and gain of gene function in plants
Erin Gilchrist and George Haughn. Briefings in Functional Genomics (2010) 9 (2): 103-110
2. Forward genetics and map-based cloning approaches. Janny L. Peters, Filip Cnudde and Tom Gerats. TRENDS in Plant Science.Vol.8 No.10 October 2003
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