Dataset Information

First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

ABSTRACT: The CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11240-021-02103-5. Key message Genome edited poinsettia plants show loss-of-function mutations in the F3’H gene introduced by CRISPR/Cas9. This is sufficient to change the bract colour from red to reddish orange.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11240-021-02103-5.

SUBMITTER: Nitarska D

PROVIDER: S-EPMC8550517 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sweet basil (Ocimum basilicum) is an economically important herb and its global production is threatened by basil downy mildew caused by the obligate biotrophic oomycete Peronospora belbahrii. Effective tools are required for functional understanding of its genes involved in synthesis of valuable secondary metabolites in essential oil and disease resistance, and breeding for varieties with improved traits. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene editing technology has revolutionized crop breeding and functional genomics. The applicability and efficacy of this genomic tool in the allotetraploid sweet basil were tested by editing a potential susceptibility (S) gene ObDMR1, the basil homolog of Arabidopsis DMR1 (Downy Mildew Resistant 1) whose mutations conferred nearly complete resistance against Arabidopsis downy mildew pathogen, Hyaloperonospora arabidopsidis. Two single guide RNAs targeting two different sites of the ObDMR1 coding sequence were designed. A total of 56 transgenic lines were obtained via Agrobacterium-mediated stable transformation. Mutational analysis of 54 T0 transgenic lines identified 92.6% lines carrying mutations at target 1 site, while a very low mutation frequency was detected at target 2 site. Deep sequencing of six T0 lines revealed various mutations at target 1 site, with a complete knockout of all alleles in one line. ObDMR1 homozygous mutant plants with some being transgene free were identified from T1 segregating populations. T2 homozygous mutant plants with 1-bp frameshift mutations exhibited a dwarf phenotype at young seedling stage. In summary, this study established a highly efficient CRISPR/Cas9-mediated gene editing system for targeted mutagenesis in sweet basil. This system has the capacity to generate complete knockout mutants in this allotetraploid species at the first generation of transgenic plants and transgene-free homozygous mutants in the second generation. The establishment of this system is expected to accelerate basil functional genomics and breeding.

Dataset Information

First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

Supplementary Information

Supplementary Information

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