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 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 feed for farmed fish in aquaculture (IRFFA, Improved Rapeseed for Fish Feed in Aquaculture), we strive to reduce glucosinolate content by knock-out of genes involved in the aliphatic glucosinolate biosynthesis and transportation pathway by a conventional 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 the conventional gel-based LI-COR TILLING and a novel NGS TILLING protocol. 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 and transporter gene BnGTR2 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 and a whole genome NGS approach. Loss of function mutations within candidate genes have been combined by crossing TILLING mutants and further backcrossed to reduce background mutation loads. Plants bearing mutations within the selected genes will be phenotyped for 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

       a.abbadi@npz-innovation.de

  • Prof. Dr. Carsten Schulz, Christian-Albrechts-Universität zu Kiel

       cschulz@tierzucht.uni-kiel.de

Additional Partners

  • Dr. Christian Obermeier, Justus-Liebig-Universität Gießen

       Christian.Obermeier@agrar.uni-giessen.de

Commercial Partners

  • Dr. Amine Abbadi, Norddeutsche Pflanzenzucht Innovation GmbH, Hohenlieth

        a.abbadi@npz-innovation.de

  • Dr. Frank Pudel, Pilot Pflanzenöltechnologie Magdeburg e.V

        pudel@ppm-magdeburg.de

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.
  • Lee, H., Chawla, H. S., Obermeier, C., Dreyer, F., Abbadi, A., & Snowdon, R. (2020). Chromosome-scale assembly of winter oilseed rape Brassica napus. Frontiers in Plant Science, 11, 496.

 

Poster presentations

  • GPZ Conference Genome Research (2019): 28.03.2019 – 29.03.2019, Hohenheim, Germany
  • 15th International Rapeseed Congress (2019): 16.06.2019 – 19.06.2019, Berlin, Germany                     
  • Kiel Plant Center–Summer Symposium (2019): 22.07.2019 – 23.02.2019, Kiel, Germany
  • International Symposium of the Society for Plant Breeding (GPZ): 11.02.2020 – 13.02.2020, Tulln, Austria

 

Financial Support

Funding has been provided by the Federal ministry for research and technology (BMBF) within the joint project IRFFA: Verbesserte Rapssaat als Fischfutter in der Aquakultur (grant number 031B0357B)

Updated: July 2020              Responsible for this webpage: Srijan Jhingan