Mutations in wheat vernalization VRN3 using CRISPR/Cas9 or EMS cause later- or no-flowering

Key message In wheat, the Vernalization 3 ( VRN3 = FLOWERING LOCUS T, FT ) is a key but not the only player for controlling the vegetative to reproductive transition. Wheat VRN3 also appears to be involved in plant and spike architecture and seed set. Abstract Wheat yield depends upon the conversion from vegetative to reproductive growth at an environmentally appropriate time. Spring wheat transitions automatically but winter wheat needs a prolonged cold period (vernalization) to transition. Using an EMS-mutagenized wheat population, we selected 88 tillering only (TO = no extension, no heading) and seven extension only (EO = slight extension, no heading) plants. Sequencing the exon regions of VRN1 and VRN3 revealed that only three TO plants had mutations in VRN1 and only seven TO plants had mutations in VRN3 , suggesting that mutations in other genes also confer the TO phenotype. In order to clarify the regulatory role of the VRN3 and VRN1 genes in the transition from vegetative to reproductive growth, CRISPR/Cas9 was used to edit the three orthologous copies (or homeologs) in each of the two genes. A role for VRN1 in wheat morphogenesis was not clearly established. In contrast, VRN3 -edited plants had phenotypes of delayed heading or no heading at all, confirming the key role of the VRN3 gene in wheat development. Different VRN3 edits resulted in different flowering times, formation of indeterminate and paired spikelets, aerial node branching and adventitious roots, indicating that the VRN3 gene may serve as a core hub not only in vegetative-reproductive transition and flowering time, but also in plant and spike architecture. This study revealed 1) the multi-functionality of the VRN3 gene in wheat reproductive development, and 2) that targeted editing of VRN3 can create new alleles for wheat breeding regarding growth habit and flowering.