TILLING is a reverse-genetics technique that allows the identification of an allelic series of induced point mutations in candidate genes in a collection of chemically mutagenized individuals or in natural plant or animal populations (EcoTILLING).
In the TILLING procedure gene segments are amplified using fluorescently-tagged primers. The forward and reverse primers to amplify the target gene of interest are labeled with specific fluorophors. The amplified PCR products are denatured and re-annealed to form heteroduplexes between the mutated amplicon and its wild-type counterpart. These heterduplexes are substrate for cleavage by a mismatch-specific nuclease. So far the cleavage products have been analyzed using polyacrilamide sequencer detection systems, which, although efficient, are not amenable to very high-throughput and automated analyses. We have developed the use of a high-throughput capillary electrophoresis system for TILLING analysis, called FLUOTILL.
The FLUOTILL platform couples the use of a capillary electrophoresis system with the automation of the entire workflow, using Tecan Freedom Evo Liquid Handling platforms.
Automated genomic DNA extraction is performed on freeze-dried leaves using a magnetic beads or column-based protocols (depending on the species) on Tecan Freedom Evo Liquid Handling Platform. Purified genomic DNA is subsequently quantified, normalized, and diluted to create working stocks in 96 well bar-coded plates.
Diluted genomic DNAs is both 8-fold and 12-fold pooled, according to the scheme below. Pooled DNAs, diluted DNAs and original DNAs are stored at -80° to create a biorepository used to carry out the TILLING analyses.
Primers for TILLING target genes are designed with CODDLE and PRIMER3. PCR is performed with or FAM-VIC or NED-PET labeled primers on pooled DNAs.
PCR amplification is followed by a denaturation-slow renaturation step and subsequent cleavage with a Mismatch Specific Nuclease.
PCR amplicons from candidate genes, cleaved with a mismatch-specific nuclease are loaded on the ABI 3730 capillary DNA analyzer.
Generated data are analyzed with the GeneMapper4.0 software. Individual candidate mutants, giving rise to the same digestion peaks both in the 8-fold and 12-fold pools (2D screening) are selected and validated via direct sequencing. Mutated nucleotides are identified using the Mutation Surveyor software.
General Tilling Strategy
Screened Tilling Populations
Barley (Morex) - TILLMORE
The entire FLUOTILL workflow was validated on a large NaN3-induced Hordeum vulgare mutant collection consisting of 5000 M3 families developed at DiSTA - University of Bologna using the 2D pooling strategy: The entire barley mutant population was screened for 11 genes and a total of 69 mutants were identified and confirmed via direct sequencing. The average number of mutants identified in the TILLMORE collection is 6 to 7 using the FLUOTILL protocol. Details of the TILLMORE collection can be found here.
Rice (Volano) - RICE-TILL
In the framework of the VALORYZA project, a TILLING rice population derived from Ethyl Methan Sulphonate (EMS) treatment of the variety Volano was created. The Volano Italian rice variety was chosen as being representative of the traditional rice quality and of relevance for ongoing breeding programs in Italy. High-throughput screening to identify mutations in genes of interest in the Volano TILLING population can be carried out at the PTP Genomics Platform using the validated FLUOTILL technology. More information can be found here
FLUOTILL Screening Service
The FLUOTILL services offered by PGP entail:
* Creation of DNA Tilling Resources: from genomic DNA extraction from TILLING populations, to the creation of 2D pools.
* Screening Tilling Populations: design of primers in selected target genes, screening of 2D TILLING pools with FLUOTILL workflow, validation of candidate mutants via direct sequencing.
In relazione a questo servizio è possibile: