Repositorium
Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA
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303
Typ / Jahr
Journal Article / 2018
Autoren
Kanazashi, Yuhei; Hirose, Aya; Takahashi, Ippei; Mikami, Masafumi; Endo, Masaki; Hirose, Sakiko; Toki, Seiichi; Kaga, Akito; Naito, Ken; Ishimoto, Masao; Abe, Jun; Yamada, Tetsuya
Abstract
Key message Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds.
Abstract The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.
Keywords
CRISPR/Cas9; Generation; Glycine max; Heritable mutation; Null-segregant; PEAPOD
Periodical
Plant cell reports
Periodical Number
3
Page range
553–563
Volume
37
DOI
10.1007/s00299-018-2251-3
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
713 |
Yamada, Tetsuya Japan |
Glycine max |
CRISPR/Cas9 PPD1/2 |
Dome-shaped trifoliate leaves, extremly twisted pods, production of few seeds SDN1 |
Basic research Basic research |