TILLING is a popular technique of reverse genetics for detection of mutation in a target gene followed by assignment of the phenotypes to the gene sequence. Point mutation is commonly induced by chemical mutagens such as EMS. Of the several techniques of reverse genetics such as RNA interference suppression and transposon tagging, Tilling rapidly gained popularity due to its amenity to automation. High throughput mutation discovery is vital for screening thousands of samples. Besides being a non-GMO approach for broadening the genetic base, it provides tools for development of markers for marker-assisted breeding for traits that are cumbersome and expensive to measure. The component of chemical mutagenesis with EMS will involve optimization of the concentration of EMS and the duration of treatment to induce point mutation while maintaining viability of the treated plant part. A number of methods have so far been utilized for detection of single nucleotide mutation. Screening of mutants with IRDye labeled primers assayed on Li-Cor platform is, however, the most well established and commonly used technique in TILLING [12]. Alternative techniques for TILLING include the use of fluorescent dye labeled primers assayed on ABI genetic analyzer.
In IITA, we are attempting to optimize the protocol for the ABI instrument. Preliminary Tilling work to discover induced and natural mutation in cassava was geared towards specific traits that are intractable using conventional methods. Adaptation of the technique to other IITA mandate crops such as yam, banana, and cowpea entails selection of target tissue/organ for mutation, and selection of similar or different target genes for tilling. Crops such as maize and soybean have numerous resources that can be easily adopted and adapted.
The prerequisite for TILLING is knowledge of nucleotide sequences of the target genes. The major IITA’s mandate crops – cassava, yam, and musa - have very limited genomic resources. To date, nucleotide sequence information for very few, largely chloroplast, genes could be found in Entrez Gene (http://www.ncbi.nlm.nih.gov/). Investigations in the past decade resulted in cloning and characterization of expressed cassava genes involved in starch, cyanogen glucoside, and carotenoid biosynthesis. Therefore, we have utilized about six 6 full gene sequences (both nuclear and chloroplast genes) and three promoter sequences of Manihot esculenta for designing primers. However, even in the absence of nucleotide sequence for the gene of interest, comparative genomics have been successfully utilized to identify candidate genes. The completion of poplar genome sequence and, more recently, that of castor bean, is expected to provide useful genetic tools for the identification of candidate genes in cassava. Besides, the ongoing cassava genome sequencing (http://www.jgi.doe.gov/sequencing/why/CSP2007/cassava.html) is anticipated to be completed soon, opening a new avenue of research in functional post-genomic studies such as TILLING.
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