Multiplex CRISPR/Cas9 Mutagenesis of BrVRN1 Delays Flowering Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

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.

In-Depth Sequence Analysis of Bread Wheat VRN1 Genes

The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

Identification of allelic combinations of the Ppd-D1, Vrn-A1, Vrn-B1 and Vrn-D1 genes in common wheat lines obtained in the National Center of Grain named after P. P. Lukyanenko

An analysis of the allelic composition of the genes determining photoperiodic sensitivity (Ppd-D1) and the need for vernalization (Vrn-A1, Vrn-B1, Vrn-D1) was carried out in 286 common wheat lines obtained in the National Center of Grain named after P. P. Lukyanenko with the use of allele-specific primers. The analyzed samples were distributed over 21 haplotypes; the dominant allele of the Ppd-D1a gene prevailed in the studied material. 123 lines of common wheat carry a combination of D-RRD alleles. The lines that can be attributed to the group of alternate wheat (R-RDR, R-RRD) were identified. All studied samples carry the recessive allele of at least one VRN1 gene.

The central role of the VERNALIZATION1 gene in the vernalization response of cereals

Many varieties of wheat (Triticum spp.) and barley (Hordeum vulgare L.) require prolonged exposure to cold during winter in order to flower (vernalization). In these cereals, vernalization-induced flowering is controlled by the VERNALIZATION1 (VRN1) gene. VRN1 is a promoter of flowering that is activated by low temperatures. VRN1 transcript levels increase gradually during vernalization, with longer cold treatments inducing higher expression levels. Elevated VRN1 expression is maintained in the shoot apex and leaves after vernalization, and the level of VRN1 expression in these organs determines how rapidly vernalized plants flower. Some alleles of VRN1 are expressed without vernalization due to deletions or insertions within the promoter or first intron of the VRN1 gene. Varieties of wheat and barley with these alleles flower without vernalization and are grown where vernalization does not occur. The first intron of the VRN1 locus has histone modifications typically associated with the maintenance of an inactive chromatin state, suggesting this region is targeted by epigenetic mechanisms that contribute to repression of VRN1 before winter. Other mechanisms are likely to act elsewhere in the VRN1 gene to mediate low-temperature induction. This review examines how understanding the mechanisms that regulate VRN1 provides insights into the biology of vernalization-induced flowering in cereals and how this will contribute to future cereal breeding strategies.