International Journal of Pure & Applied Bioscience (IJPAB)
Year : 2017, Volume : 5, Issue : 5
First page : (669) Last page : (679)
Article doi: http://dx.doi.org/10.18782/2320-7051.2798
Tilling: Is Effective Tool for Mutant Gene Detection in Crop Improvement
Baldeep Singh1*, Geeta Boken2 and Geetika Mehta3
1Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana- 141004
2Department of Genetics and Plant Breeding, CCS Haryana Agricultural University, Hisar- 125004
3Indian Institute of Wheat And Barley Research,Karnal, Haryana 132001
*Corresponding Author E-mail: baldeep.singh55@gmail.com
Received: 5.04.2017 | Revised: 12.05.2017 | Accepted: 23.05.2017
ABSTRACT
Targeting Induced Local Lesions IN Genomes (TILLING) is a functional genomics tool that comprise heteroduplex analysis to detect which organisms in a population carry single nucleotide mutations in specific genes. TILLING is a general reverse genetic strategy that is high in throughput, low in cost, applicable to most organisms and works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. It remains a dominant non-transgenic method for obtaining mutations in known genes and next-generation sequencing (NGS) to the process. NGS will ultimately lead to TILLING. Mutagens like, Ethylmethanesulfonate (EMS), cause stable point mutations and thus produce an allelic series of truncation and missense changes that can deliver a range of phenotypes. Tilling allocates the rapid and cost-effective detection of induced point mutations in populations of physical/chemically mutagenized individuals. EMS is produce G/C to A/T transition by alkylating the G residues and the alkylated G resides to base pair with T instead of pairing with C. In TILLING parental DNA sequence whereas in EcoTILLING genomic DNA sequence is used to identify the mutations created using endonuclease (CEL I). Genes are amplified by PCR using pooled genomic DNA from several individuals as a template. Following denaturation and renaturation of the amplified DNA, heteroduplexes form individuals with wild type and mutant sequence are both present in the pool. The heteroduplexes can be detected by cleavage with different endonuclease and resolution of the resulting fragments on a sequencing gel. TILLING can also be used to detect naturally occurring single SNP’s in genes among accessions, varieties, ecotypes or cultivars. The protocols developed for TILLING have been adapted for the discovery of natural nucleotide assortment, a method termed EcoTILLING. EcoTILLING is an extension of TILLING. Current advantages of TILLING have made it an appropriate tool for the detection of both natural and induced variation in several plant species. Here, we review recent headway in this technology for the researchers of plant mutation analysis and genomics era.
Keywords: Reverse genetics, Mutagenesis, NGS, TILLING, EcoTILLING.
Full Text : PDF; Journal doi : http://dx.doi.org/10.18782
Cite this article: Singh, B., Boken, G. and Mehta, G., Tilling: Is Effective Tool for Mutant Gene Detection in Crop Improvement, Int. J. Pure App. Biosci.5(5): 669-679 (2017). doi: http://dx.doi.org/10.18782/2320-7051.2798
