CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape ( Brassica napus ) .

Braatz, Janina, Harloff, Hans-Joachim, Mascher, Martin, Stein, Nils, Himmelbach, Axel and Jung, Christian (2017) CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape ( Brassica napus ) . Plant Physiology, 174 (2). pp. 935-942. DOI 10.1104/pp.17.00426.

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Abstract

In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape (Brassica napus) with a CRISPR-Cas9 construct targeting two ALCATRAZ (ALC) homoeologs. ALC is involved in valve margin development and, thus, contributes to seed shattering from mature fruits. Knocking out ALC would increase shatter resistance to avoid seed loss during mechanical harvest. We obtained a transgenic T1 plant with four alc mutant alleles by the use of a single target sequence. All mutations were stably inherited to the T2 progeny. The T2 generation was devoid of any wild-type alleles, proving that the underlying T1 was a nonchimeric double heterozygote. T-DNA and ALC loci were not linked, as indicated by random segregation in the T2 generation. Hence, we could select double mutants lacking the T-DNA already in the first offspring generation. However, whole-genome sequencing data revealed at least five independent insertions of vector backbone sequences. We did not detect any off-target effects in two genome regions homologous to the target sequence. The simultaneous alteration of multiple homoeologs by CRISPR-Cas9 mutagenesis without any background mutations will offer new opportunities for using mutant genotypes in rapeseed breeding.

Document Type: Article
Research affiliation: Kiel University > Kiel Marine Science
OceanRep > The Future Ocean - Cluster of Excellence
Kiel University
Refereed: Yes
Open Access Journal?: No
DOI etc.: 10.1104/pp.17.00426
ISSN: 0032-0889
Projects: Future Ocean
Date Deposited: 19 Dec 2017 11:08
Last Modified: 15 Apr 2019 14:38
URI: http://oceanrep.geomar.de/id/eprint/40892

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