Plant Breeding Institute

Functional analysis and mutagenesis of glucosinolate synthesis genes for breeding oilseed rape (B. napus) with lower glucosinolate content

Background

Glucosinolates (GSL) are a class of heterogeneous secondary metabolites specific to Brassicales. They are natural organic compounds containing sulfur and nitrogen that are synthesized from glucose and amino acids. Depending upon the amino acid constituting the structure, they are classified into aliphatic, aromatic and indolic GSLs. Owing to their anti-nutritive and toxic nature, GSLs play a major role in defensive mechanisms against herbivory. Glucosinolates are precursors of mustard oils; secondary by-products like isothiocyanates, thiocyanates and nitriles which are generated after the enzymatic cleavage by the endogenous myrosinase. Some are toxic when consumed in higher concentrations in humans and animals and cause goiter formation or renal and hepatic damage among other anti-nutritive effects. From a diversity panel of ~120 different compounds reported from Arabidopsis, 15 major glucosinolate types have been identified in B.napus seeds. Utilization of rapeseed meal (containing ~40% protein) as a source of animal feed is restricted due to these toxic and anti-nutritive compounds that even in canola quality rapeseed (00) can still reach concentrations of 25 µmol/g in seeds.

As part of a larger project to develop rapeseed meal as fish feed for farmed fish in aquaculture (IRFFA, Improved Rapeseed for Fish Feed in Aquaculture), we strive to reduce glucosinolate content by knock-out approaches of genes of the glucosinolate biosynthesis pathway by TILLING and the CRISPR-Cas9 approach.

Objectives

The aim of the project is to identify loss of function mutations i.e. non-sense, splice site and missense mutations within GSL biosynthesis and transporter genes in an existing Express617 winter rapeseed TILLING population. This will be achieved by LI-COR TILLING and a novel NGS TILLING protocol. Candidate genes will be selected according to low copy number and tissue-specific expression analysis in leaves and seeds at different growth stages. Putative loss of function mutants for candidate genes will be combined by crossing and screened for reduced GSL content.

Results

Via in silico analysis using Brassica databases (BRAD) and The Arabidopsis Information Resource (TAIR), homoeologs for two glucosinolate biosynthesis genes and one transporter gene were identified in B. napus using the reference rapeseed genome (Genoscope). Biosynthesis genes BnMYB28 and BnCYP79F1 with 2 paralogs each were selected as appropriate candidates for TILLING. Expression profiles of the candidates in seeds and leaves of winter type oilseed rape Express617 have shown differential expression during seed maturation. Candidate genes have been selected according to low copy numbers and tissue-specific expression analyses in leaves and seeds at 15, 25, 35 and 45 days after pollination. Putative functional mutations have been identified for each of the candidate paralogs following a conventional TILLING by LI-COR approach. Loss of function mutations within candidate genes will be combined by crossing TILLING mutants and be phenotyped for aliphatic GSL content in leaves and seeds. With successful down-regulation of genes involved in the aliphatic biosynthesis pathway, a significant reduction in the aliphatic GSL is expected.

Project team

M.Sc. Srijan Jhingan
Dr.  Hans-Joachim Harloff
Prof. Dr. Christian Jung

Scientific Partners

Dr. Amine Abbadi (Co-ordination), NPZ Innovation GmbH
Prof. Dr. Carsten Schulz, Christian-Albrechts-Universität zu Kiel

Additional Partners

Prof. Dr. Rod Snowdon, Dr. Christian Obermeier, Justus-Liebig-Universität Gießen

Commercial Partners

Dr. Amine Abbadi, Norddeutsche Pflanzenzucht Innovation GmbH, Hohenlieth
Dr. Frank Pudel, Pilot Pflanzenöltechnologie Magdeburg e.V

Publications

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

Braatz, J., Harloff, H. J., Mascher, M., Stein, N., Himmelbach, A., & Jung, C. (2017). CRISPR-Cas9 induced mutations in polyploid oilseed rape. Plant physiology, pp-00426.

Financial Support

Funding has been provided by the Federal ministry for research and technology (BMBF) within the joint project IRFFA.

 

Updated: June 2019                     Responsible for this webpage: Srijan Jhingan