Background

Seed oil content is determined by the balance between seed fatty acids synthesis and degradation. To date, many attempts have been made to increase the oil content in rapeseed via improving seed fatty acid biosynthesis. On the other hand, it has been reported that after an increase during the first 35 days after pollination, rapeseed oil content is declining to a small extent at the late stage of seed development until maturation, indicating that degradation processes might play a role (Fowler and Downey 1970; Kelly et al. 2013). In Arabidopsis, five Seed Fatty Acid Reducer (SFAR) genes have been identified, which belong to the GDSL lipase/esterase family and have been shown to be involved in the seed fatty acid reduction in mature seeds (Chen et al. 2012).

Due to the close evolutionary relationship of Arabidopsis and Brassica napus, we started to investigate the effect of corresponding homologous gene copies on the oil content in rapeseed. In this project, we follow two knock-out approaches using classical TILLING in an existing EMS (ethyl methanesulfonate) mutated winter rapeseed population and the CRISPR/Cas9 technique.

Objectives

The objectives of this project are to identify missense and nonsense mutations within BnSFAR genes by TILLING and produce homozygous mutant lines. Single mutants are crossed to obtain double mutants and backcrosses are performed to reduce the mutational load of the mutant lines. For comparison, the CRISPR/Cas9 system is used to obtain knock-out mutants without mutation background and for a simultaneous knock-out of more than one candidate gene. Mutants will be analyzed phenotypically for the oil content, fatty acid profile and lipase activity in the developing seeds. In addition, expression profiles and seedling performance will be monitored

Results

By in silico analysis, 12 homologous genes were identified in B. napus genome for 5 known Arabidopsis SFAR genes. Based on the relative expression values, 6 paralogs were selected for mutation screening by TILLING. Single mutants are combined to produce double mutants expecting pyramiding of mutated bnsfar genes could increase the seed oil content significantly compared to the single mutants.

The CRISPR/Cas9 system was employed to generate mutations in all 12 paralogs in each BnSFAR subfamily using a common subfamily-specific target region. Agrobacterium-mediated hypocotyl transformation was used for plant transformation. Until now, transformation efficiency is between 0.1- 1.1%. We identified InDels mutations in T₂ plants for some of BnSFAR paralogs. Oil content and fatty acid composition were measured in the T₃ and T₄ seeds after having fixed the genotypes and revealed that oil content is significantly higher in mutants in comparison to wild type.

Project team

M.Sc Nirosha L. Karunarathna
Dr. Hans-Joachim Harloff
Prof. Christian Jung

Scientific Partners

Professor Lixi Jiang, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

Publications

Harloff HJ, Lemcke S, Mittasch J, Frolov A, Wu JG, Dreyer F, Leckband G, Jung C (2012) A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet 124: 957-969

Posters and Oral Presentations

Karunarathna N.L., Harloff, H.-J., Jung, C (2019) Knock-out of SEED FATTY ACID REDUCER4 by CRISPR-Cas9 increases seed oil content in rapeseed (Brassica napus), Kiel Plant Science symposium 17.01.2019, Kiel. Oral presentation

Karunarathna N.L., Wang H.,  Harloff, H.-J., Jiang L., Jung, C. (2018). Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (Brassica napus) with higher oil content   GPBC2018, Wernigerode, 28.02-02.03.2018. Poster Presentation

Karunarathna N.L., Wang H.,  Harloff, H.-J., Jiang L., Jung, C. (2018). Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (Brassica napus) with higher oil content   III. International PP1530 Symposium: Genetic Variation of Flowering Time Genes and Applications for Crop Improvement, Kiel, 14-16.02.2018. Poster Presentation

Karunarathna N.L., Wang H.,  Harloff, H.-J., Jiang L., Jung, C. (2017). Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (Brassica napus) with higher oil content   Botanikertagung 2017, Kiel, 17.-21.09.2017. Poster Presentation

Karunarathna N.L., Wang H.,  Harloff, H.-J., Jiang L., Jung, C. (2017). Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (Brassica napus) with higher oil content   PP1530 International Workshop: ‘Genome Engineering’, Gatersleben, 22.-23.06.2017. Poster Presentation

Financial Support (three years)

The project is a joint Sino-German research project.

It is funded by the Sino-German Center (GZ1099) and the Deutsche Forschungsgemeinschaft under grant No. DFG JU 205/25-1

Updated: 29.05.2019         Responsible for this webpage: Nirosha L. Karunarathna