CRISPR/Cas9-mediated Editing of SlMYB12 for Breeding Pink-fruited Tomato

doi:10.7668/hbnxb.20195300

Abstract

Abstract:

In order to accelerate the breeding of pink-tomato varieties,a rapid breeding strategy was used to generate pink-fruited tomato material by CRISPR/Cas9-mediated gene editing of SlMYB12.Two adjacent target sites within the first exon of SlMYB12 were selected to construct the CRISPR/Cas9 binary vector, and then the vector was introduced into red-fruited inbred line R18-10C through Agrobacterium-mediated transformation. The homozygous mutants without exogenous Cas9 were screened using specific primers and their agronomic traits and fruit nutritional quality were analyzed. Sequencing results showed that three homozygous mutants of different mutation types were obtained and all of which were frame shift mutations caused by base deletion. Compared with wild-type red fruit tomatoes, SlMYB12-edited plants grew and developed normally,and there were no significant difference in plant height,single-fruit weight,total yield per plant,fruit total soluble solid content and lycopene level,but the mature fruit showed pink and the flavonoid naringenin chalcone(NarCh)content of tomato peels was significantly reduced in MYB12-edited plants.In summary,the procedure for the generation of pink-fruited tomato plants through CRISPR/Cas9-mediated targeted mutagenesis of SlMYB12 was set up and new pink-fruited tomato germplasms with stable inheritance were obtained.

Key words: Tomato, Pink fruit, SlMYB12, CRISPR/Cas9, Gene editing

SU Xiaomei, LYU Hongjun, LIU Shumei, LIANG Zengwen, CHEN Qian, HOU Lixia. CRISPR/Cas9-mediated Editing of SlMYB12 for Breeding Pink-fruited Tomato[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 27-32. doi: 10.7668/hbnxb.20195300.

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Figures/Tables 4

Fig.1 Construction of CRISPR/Cas9-mediated editing vector of SlMYB12 A.Schematic illustration of the two sgRNAs targeting the MYB12 coding sequence; B.Schematic illustration of T-DNA region of the CRISPR/Cas9 vector with two target sites.

Fig.2 Identification and screening of CRISPR/Cas9-MYB12 plants A.Screening of Cas9-free CRISPR/Cas9-MYB12 plants:1—24.Partial T₁ plants;B and C.Sequence-based genotyping of gene-edited T₁ homozygous plants.

Fig.3 Fruits and fruit peels of WT and three Cas9-free CR-MYB12 homozygous T₂ plants

Fig.4 Determination of agronomic traits and fruit substance content of WT and three Cas9-free CR-MYB12 homozygous T₂ plants Different letters indicate significant difference(P<0.05).

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