A rich TILLING resource for studying gene function in Brassica rapa

Area of Science:

• Plant Science
• Genetics
• Genomics

Background:

• Brassicaceae family includes important crops like Brassica napus and model plant Arabidopsis thaliana.
• Brassica species are closely related to Arabidopsis, allowing knowledge transfer for crop improvement.
• Studying gene function directly in diploid Brassica rapa is efficient due to potential gene redundancy in amphidiploid Brassicas.

Purpose of the Study:

• To develop the first ethyl methane sulfonate (EMS) TILLING population in the diploid Brassica species, B. rapa.
• To create a valuable resource for reverse genetics studies in B. rapa, aiding oilseed rape crop improvement.
• To establish a densely mutated diploid organism for efficient gene function elucidation.

Main Methods:

• Ethyl methane sulfonate (EMS) treatment of B. rapa seeds (R-o-18 genotype) to create a TILLING population.
• DNA isolation from 9,216 M2 plants and pooling for TILLING platform.
• Analysis of six genes to determine mutation density and screening efficiency.

Main Results:

• A high mutation density of approximately 1 mutation per 60 kb was achieved in the B. rapa TILLING population.
• Screening a 1 kb amplicon in 3072 M2 plants yielded an average of 68 mutations, with a 97% probability of stop-codon mutations.
• Each plant contains an average of 10,000 mutations, with predicted mutations in half of the genome's GC base pairs.

Conclusions:

• The first EMS TILLING resource in the diploid Brassica species, B. rapa, has been successfully developed.
• This B. rapa TILLING population exhibits the highest mutation density reported for a diploid organism.
• The resource is publicly accessible via the RevGenUK reverse genetics platform for research use.