Lehrstuhl für Pflanzenzüchtung

Functional analysis and mutagenesis of GDSL genes for breeding oilseed rape (B. napus) with higher oil content

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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 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 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, for 5 known Arabidopsis SFAR genes 12 homologous genes were identified in B. napus. Paralog copies were classified into subgroups in alignments using structural homologies on DNA level as well as in the five conserved protein domains of the GDSL type BnSFAR proteins.

Based on the relative expression values, 6 paralogs out of 12 paralogs were selected for mutation screening by TILLING. Screening of EMS mutated winter rapeseed Express 617 population allowed to identify nonsense, missense, splice site, silence and UTR mutations. For each paralog, prominent mutants containing nonsense, splice site and missense mutations were selected. In parallel to single mutant analysis, we will combine single mutants discovered by TILLING to produce double mutants to increase the seed oil content by pyramiding of mutated bnsfar genes.

The CRISPR/Cas9 system was employed to generate mutations in all 12 paralogs in each BnSFAR subfamily using a common subfamily-specific target region aiming to increase seed oil content in rapeseed. Agrobacterium-mediated hypocotyl transformation was used for three rapeseed genotypes, Haydn, RS306 and Express 617. Selection with phosphinothricin resulted in putative transgenic plants. DNA will be isolated from these T1 plants and Sanger sequencing will be done to confirm the gene editing. Oil content and fatty acid composition will be measured in the T3 seeds after having fixed the genotypes in T2.

.Project team

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

Scientific Partners

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

Commercial Partners

Norddeutsche Pflanzenzucht Innovation GmbH, Hohenlieth

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

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-May-2017         Responsible for this webpage: Nirosha L. Karunarathna