The TILLING Platform

 

Genomic DNA from the plants of the M₂ population was isolated by the John Innes Genome Laboratory (JGL).  After isolation, the DNA was normalised and used to set up a TILLING platform in a 96-well format with pools of eight gDNA samples per well. This means that each 96-well plate contained DNA from 768 M₂ plants. Twelve plates have been made, making an entire population of:

 

768 M₂ plants per plate x 12 plates = 9,216 M₂ plants

 

Mutations are detected by Cel1 digests run on an ABI3730 sequencing machine. After detecting a Cel1 product in the eight-pool (confirmed using fluorescent primers from both ends), individual gDNA samples are sequenced to identify the exact plant harbouring the mutation.

 

Cel1 digestion

Sequence confirmation

    F wt

    F sample

    R sample

    R wt

 

We perform the TILLING assays on ~1-kb amplicons and have done a number of ‘TILLs’ already. The statistics of some of those are summarised below.  These data show a remarkably high mutation load with an average of ~240 mutations per kb (amplicon) if the entire population were screened (9,216 M₂ plants).  In our experience, screening four plates (3,072 M₂ plants) usually provides a sufficient number of useful mutations, including stop codon mutations and amino acid changes.

 

Establishment of the resource was funded by the AdVaB project and mutants in genes-of-interests can be ordered by the community through RevGenUK. Please go to the RevGenUK website to enquire about the sequence information which is required.

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Norwich Research Park,
Colney, Norwich NR4 7UH, UK