The VERNALIZATION1 (VRN1) gene is a crucial transcriptional repressor involved in triggering the transition to flowering in response to prolonged cold. To develop Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants with delayed flowering time, we designed a multiplex CRISPR/Cas9 platform that allows the co-expression of four sgRNAs targeting different regions of the endogenous BrVRN1 gene delivered via a single binary vector built using the Golden Gate cloning system. DNA sequencing analysis revealed site-directed mutations at two target sites: gRNA1 and gRNA2. T₁ mutant plants with a 1-bp insertion in BrVRN1 exhibited late flowering after the vernalization. Additionally, we identified ‘transgene-free’ BrVRN1 mutant plants without any transgenic elements from the GE₁ (gene-editing 1) and GE₂ generations. All GE₂ mutant plants contained successful edits in two out of three BrVRN1 orthologs and displayed delayed flowering time. In GE₂ mutant plants, the floral repressor gene FLC1 was expressed during vernalization; but the floral integrator gene FT was not expressed after vernalization. Taken together, our data indicate that the BrVRN1 genes act as negative regulators of FLC1 expression during vernalization in Chinese cabbage, raising the possibility that the ‘transgene-free’ mutants of BrVRN1 developed in this study may serve as useful genetic resources for crop improvement with respect to flowering time regulation.

The emergence and expression of the YABBY gene family (YGF) coincided with the evolution of leaves in seed plants, and was integral to the early evidence of lamina followed by reproductive development. YGF contains six subclasses, i.e., CRC, INO, FIL, YAB2, YAB3, and YAB5. This study aims to extract the genome sequences of the YGF in Bienertia sinuspersici, an important model plant for single-cell C₄ (SCC₄), non-Kranz photosynthesis. A comparative genomic analysis was undertaken with Vitis vinefera, Arabidopsis thaliana, Brassica rapa, and Chenopodium quinoa. Six copies of YGF were present in B. sinuspersici and A. thaliana with a single copy of each YGF subgroup. V. vinefera possessed seven copies of YGF with duplicates in FIL and YAB2 subgroups, but no YAB3. B. rapa and C. quinoa after whole genome duplication contained additional copies of YGF. The gene structure and conserved motifs were analyzed among the YGF. In addition, the relative quantification of YGF was analyzed in the leaves, reproductive developmental stages such as the bud, and the pre-anthesis and anthesis stages in B. sinuspersici, A. thaliana, and B. rapa. CRC and INO possessed conserved floral-specific expression. Temporal and perpetual changes in the expression of YGF orthologs were observed in the leaves and reproductive developmental stages. The results of this study provide an overview of YGF evolution, copy number, and its differential expression in B. sinuspersici. Further studies are required to shed light on the roles of YABBY genes in the evolution of SCC₄ plants and their distinct physiologies.