Background                                                                                                

Seed oil content (SOC) is determined by the balance between seed fatty acid synthesis and degradation. 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 lipase activity might play a role. 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.

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 followed two knock-out approaches using classical TILLING in an existing EMS (ethyl methanesulfonate) mutated winter rapeseed population and the CRISPR-Cas9 technique.

Objectives

In this study, the main focus was to increase seed oil content by knocking out genes involved in the lipid degradation pathway, and their phenotypic characterization under greenhouse experiments. Furthermore, we aimed to reduce the background mutation load caused by EMS-induced mutagenesis by implementing marker-assisted genomic selection and rapid cycle breeding of rapeseed.

Results

We selected five known SFAR genes that function to decrease SOC in Arabidopsis and searched for homologous in oilseed rape. After quantification of SFAR gene expression using RNA-seq and RT-qPCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter oilseed rape. We were able to increase SOC significantly in mutants by 14.5% when compared to the wildtypes.

The background mutation load should be eliminated with repeated backcrossing. We backcrossed EMS mutants with a fast flowering spring variety to reduce background mutation and to simultaneously accelerate generation time. We used 15K Illumina Infinium^TM oilseed rape SNP-array for genomic background selection, and we were able to identify mutant plants with ~85% of the recurrent parent’s genome in the BC₁ generation. Currently, we carry out the mutant gene pyramiding by combining the individual mutants with the highest recurrent parent’s genome.

Project Team

Dr. 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., Wang H., Harloff H.-J., Jiang L., Jung C. (2020), Elevating seed oil content in a polypoid crop by induced mutations in SEED FATTY ACID REDUCER genes, International Symposium of the Society for Plant Breeding (GPZ), Tulln, Austria, 11-13.02.2020. Oral presentation

Karunarathna N.L., Harloff H.-J., Jung C., (2019), Gene knock-out by CRISPR-Cas9 and EMS-induced point mutations on SEED FATTY ACID REDUCERS increase the seed oil content in rapeseed (Brassica napus), International Rapeseed Conference, Berlin, 16-19.06. 2019. Oral presentation

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: 07.07.2020         Responsible for this webpage: Nirosha L. Karunarathna