Harue Shinoyama, Hiroaki Ichikawa, Ayako Nishizawa-Yokoi, Mikhail Skaptsov & Seiichi Toki

Scientific Reports volume 10, Article number: 16165 (2020) - Published: 30 September 2020

Genome editing has become one of the key technologies for plant breeding. However, in polyploid species such as chrysanthemum, knockout of all loci of multiple genes is needed to eliminate functional redundancies. We identified six cDNAs for the CmDMC1 genes involved in meiotic homologous recombination in chrysanthemum. Since all six cDNAs harbored a homologous core region, simultaneous knockout via TALEN-mediated genome editing should be possible. We isolated the CmDMC1 loci corresponding to the six cDNAs and constructed a TALEN-expression vector bearing a CmDMC1 target site containing the homologous core region. After transforming two chrysanthemum cultivars with the TALEN-expression vector, seven lines exhibited disruption of all six CmDMC1 loci at the target site as well as stable male and female sterility at 10–30 °C. This strategy to produce completely sterile plants could be widely applicable to prevent the risk of transgene flow from transgenic plants to their wild relatives.

See: https://www.nature.com/articles/s41598-020-72356-1

Figure 2: Structure of binary vector pBIK201DMC-TAL for disruption of six CmDMC1 genes in chrysanthemum. RB, right border; LB, left border; Pmas201, bidirectional promoter cassette from mannopine synthase 1′ and 2′ (mas1′-2′) genes; T35S, Cauliflower mosaic virus 35S terminator; Tnos, nopaline synthase terminator; nptII, neomycin phosphotransferase II gene for the selection of transgenic plants; TAL-L and TAL-R, the TALEN pair targeting CmDMC1 genes; Fok I, gene encoding a restriction enzyme. T2A, encoding a peptide for self-cleavage and ribosome skipping69. Red bar indicates the nptII-specific probe (about 800 bp) for Southern blotting in Supplementary Fig. S3.