Fruit spine density is an important commercial trait for cucumber (Cucumis sativus L.). Most North China-type cucumbers that are grown over large areas have a dense-spine phenotype, which directly affects the appearance quality, storage, and transportation of the fruits. Here, we isolated a novel few spines mutant (fs2) from the wild-type (WT) inbred line WD1, a North China-type cucumber with high density fruit spines, by an ethyl methanesulfonate (EMS) mutagenesis treatment. Genetic analysis revealed that the phenotype of fs2 is controlled by a single recessive nuclear gene. We fine-mapped the fs2 locus using F2 and BC1 populations (1,802 and 420 individuals, respectively), which showed that the candidate gene of FS2 (Csa4G652850) encodes an ARID-HMG transcription factor containing an AT-rich interaction domain (ARID) and a high mobility group box domain (HMG). One SNP (C to T) and one InDel (a 40-bp deletion) in the coding region of FS2 result in amino acid variation and premature translation termination in the fs2 mutant, respectively. FS2 was found to be highly expressed in the apical buds and young ovaries. In addition, experiments suggest that FS2 participates in the regulation of fruit spine initiation by activating the expression of the Tril gene in cucumber. This work provides not only an important reference for understanding the molecular mechanisms of fruit spine development but also an important resource for fruit appearance quality breeding in cucumber.
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The improvement and innovation of screening markers contributes to the development of transgenic technology, among which the visual screening markers are widely modified for better effect. Recent studies revealed that an enhanced Yellow Green Fluorescent like Protein (eYGFPuv (GFPuv)) obtained by mutation can emit strong and stable green fluorescence under 365 nm UV light irradiation and be easily observed. Constructing the gene editing vector with GFPuv fluorescence screening marker and carrying out experiment application and verifications in potato genetic transformation will provide technical support for the screening of positive transgenic plants in potato transformation, and lay the foundation for using genome editing technology to create potato male sterile lines in the future.
By using homologous recombination, the GFPuv expression framework and gene editing element Cas9_sgRNA were successively recombined into pCAMBIA2300 vector, and then with this new designed vector the Agrobacterium-mediated transient expression assay was conducted in tobacco plants. Six editing vectors with potato anther development conservative genes were constructed using this modified vector. The A. rhizogenes strains Ar qual and MSU440 harbouring these vectors were transformed into the potato stem segments respectively, and then the A. rhizogenes-induced hairy roots with green fluorescence were observed and counted under the portable UV lamp. The transformation efficiency and editing efficiency of these vectors were analyzed using hairy root transformation system in two different potato genotypes. In the end, the modified vectors were applied to produce transformed potato plants with modifications on target genes.
A novel gene editing vector pCAMBIA2300MGFPuv-sgRNACas harbouring a GFPuv fluorescence marker was successfully constructed, and the transient transformation in tobacco plants confirmed that the GFPuv expression framework was expressed successfully. The hairy roots with green fluorescence were screened after the transformation with two kinds of A. rhizogenes, and an additional supplement of kanamycin (Kan) significantly increased the proportion of positive fluorescent roots. Although the transformation rates of the two strains were not significantly different, the hairy roots of MSU440 formed faster. Furthermore, the transformation rates and editing rates of editing vectors for six potato anther development conservative genes in two different potato genotypes were the same, but the editing rates of six target sites differed significantly. Potato genetic transformation using the modified vector confirmed that GFPuv fluorescence could be used for the screening of transgenic callus and plants in potato.
The hairy root transformation system mediated by A. rhizogenes is an essential approach to verifying the efficiency of gene editing, and GFPuv fluorescence can be used in the screening of transgenic plants in potato transformation.
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