Plant Breeding Institute

Improving Oilseed rape by Cas9-mediated genome editing

Background 

Rapeseed is one of the most important oil crops worldwide and achieving high yields has always been the major goal in rapeseed production. The multilocular silique trait has a great potential for the development of high yield varieties of Brassica. Mutation in the CLAVATA signaling pathway genes already showed a promising phenotype in rapeseed. Another major issue in rapeseed cultivation is the pre-harvest seed loss. The fruits, so called siliques, dry out at maturity and become very fragile. Bad weather conditions as strong winds or hail can cause the siliques to break and shed the seeds. This does not only result in yield loss but produces volunteer plants in the next season resulting in quality reduction of the harvest. Knock-out mutations of ALCATRAZ (ALC) produced shatter resistant rapeseed by EMS mutagenesis and the CRISPR-Cas9 system (Braatz et al. 2017, 2018). Another trait of interest is the high amount of glucosinolate content. Glucosinolates are sulfur-containing secondary metabolites, which are common for Brassicas. The effects of these compounds on plant-pest interactions, human health after ingestion, and uptake of rapeseed-based feed by animals are complex. Although breeding has reduced the level of glucosinolates to 8–15 μmol/g in the seeds of modern 00-quality oilseed rape varieties, this amount is still too high for animal feed or human nutrition.

The CRISPR-Cas9 system has recently emerged as an efficient and versatile tool for genome editing in various organisms. However, editing efficiency and use of multiple targets are often limited by the used vector system. A general strategy and platform for precise processing and efficient production of numerous gRNAs using a polycistronic tRNA-processing system would be an advantageous tool in a polyploid crop species like rapeseed.

Objectives

By knocking out of redundant genes in parallel, we are aiming to produce rapeseed with an increased number of seed chambers, which has a potential to increase seed yield. At the same time, we will target the glucosinolate biosynthesis pathway to reduce antinutritive compounds in rapeseed products. We will apply a vector construct, which allows simultaneous expression of multiple sgRNAs to knock out gene families in rapeseed. Moreover, we expect that the polycistronic tRNA-gRNA molecules are processed by plant innate RNases and yield functional sgRNAs for targeted mutagenesis. We will also test the agronomic performance of two classes of mutants resulting in reduced seed shattering, one obtained by EMS mutagenesis and the other by Cas9-mediated mutagenesis. We expect that CRISPR-Cas modified plants are as fertile and productive as wild type plants while EMS mutants suffer from many background mutations.

Results

We are targeting BnCLV1, BnCLV2 and BnCRN as candidate genes for the multilocular trait  and BnCYP79F and BnMYB28 for the reduction of glucosinolates. Via in silico analysis using Brassica databases (BRAD) and The Arabidopsis Information Resource (TAIR), homoeologs for the candidate genes were identified in B. napus using the reference rapeseed genome (Genoscope). sgRNAs were designed for the candidate genes and  have been cloned into transformation vector. Agrobacterium mediated transformation is going on. Crosses to generate a comparable genetic background in BnALC EMS and CRISPR mutants by a novel speed breeding protocol are underway and a BC1 generation has been produced. Currently, we are genotyping BC1 plants to select double heterozygous mutants . Furthermore, we are applying  genomic background selection to identify BC1 plants with a high share of the recipient genome.

Project team

Dr. Tahmina Islam

Dr. Hans-Joachim Harloff

Prof. Dr. Christian Jung

 

Scientific Partners

Prof. Dr. rer. nat. Wolfgang Bilger, Ökophysiologie der Pflanzen, University of Kiel

Publications

Braatz J, Harloff H-J, Jung C (2018) EMS-induced point mutations in ALCATRAZ homoeologs increase silique shatter resistance of oilseed rape (Brassica napus). Euphytica 214:29 doi: 10.1007/s10681-018-2113-7

Braatz J, Harloff H-J, Mascher M, Stein N, Himmelbach A, Jung C (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol 174(2):935-942. doi:10.1104/pp.17.00426

Financial Support

Funding has been provided by the Bundesministerium für Bildung und Forschung.

 

Updated:30.06.2020            Responsible for this webpage: T. Islam