TILLING (Targeting Induced Local Lesions IN Genomes) is a new, effective tool of reverse genetics to produce and identify loss or gain of function mutants. As with classical mutation research the subject of TILLING is a mutant population. In classical studies the whole mutant population has been phenotyped in time and labour consuming observation experiments in order to identify single mutant plants for certain traits.

In TILLING, however, the mutant population is first genotyped using high throughput technology in order to identify mutants with changes within certain candidate gene sequences. Only such mutants are subsequently characterized in observation experiments allowing a much higher throughput compared to classical studies. TILLING not only allows studying gene function but it is also a method to obtain loss or gain of function genotypes of agronomical importance without production of genetically modified organisms.

Objectives

The objectives of our research are (i) to create a sugar beet (Beta vulgaris) mutant population (ii) to establish a TILLING platform for high throughput detection of mutants with point mutations in candidate sequences for flowering regulation and (iii) to study the phenotype of these mutants.

Results

An EMS mutant collection was produced to identify loss-of-function mutants in a bolting sugar beet genotype. The M0 plants have been treated with different EMS concentrations for four different times of incubation in EMS to cover a wide range of mutagen concentrations. Approximately 70,000 M2 plants that originated from more than 3,000 selfed M1 plants were grown, and 5 independent non-bolting plants have been identified, suggesting effective EMS mutagenesis.

For the TILLING project, approx. 11,000 M2 plants have been grown and selfed in the field. Leaf material has been collected and DNA extraction is presently performed using a 96-well format plant DNA extraction kit. A TILLING platform for sugar beet including an efficient pooling strategy of DNA and an optimized TILLING protocol for heteroduplex detection on LI-COR DNA-Analyzer will be established. Together with our project partners a database for TILLING-primer design, submission of screening requests, and documentation of results is constructed.

Project team

Friedrich Kopisch-Obuch
Sebastian Frerichmann
Verna Kowalewski
Monika Bruisch
Birgit Defant
Christian Jung

Last Updated ( Monday, 27 October 2008 )