TILLING (Targetted Local Lesions IN Genomes) offers a route for carrying out reverse genetics without the need for extensive genomic information.
A TILLING approach to reverse genetics requires two strategic resources; a TILLING population of seed derived from individual mutagenised plants, and a set of high quality DNA samples produced from the individual parent plants of the seed population organised into pools for molecular screening.
Development of a TILLING population for lettuce is timely as new genomic resources are rapidly becoming available; combining a TILLING approach with microarray/gene expression and QTL studies will facilitate research to identify genes underlying QTL for important traits for sustainability and product quality.
We derived a TILLING population from the cv. Saladin which is one of the parents of the WHRI lettuce mapping population and a mapping population developed by Prof. Richard Michelmore at UC Davis. Seed were treated with the chemical mutagen ethyl-methyl sulphonate (EMS), EMS is a mutagen that induces point mutations in DNA.
The plants that survived the EMS treatment were grown up and taken through two rounds of selfing.
Four thousand M2 plants were grown in 2008 and duplicate leaf samples were taken from each plant and freeze-dried for DNA extraction.
Fertile plants surviving to maturity were bagged for self pollination and seed were collected and dried.
We have collected M3 seed from 3240 lines.
A total of 3,800 DNA samples were extracted in 40 x 96-well plates.
Gel analysis showed that the DNA was of high molecular weight with no evidence of shearing or degradation.
The DNA samples were placed in the Warwick HRI freezer archive facility for secure long-term storage.
Assessment of the DNA mutation load is being carried out by BBSRC ReVGenUK at John Innes Centre.
Enquires relating to accession details and seed requests should be made to Graham Teakle.