Use of TILLING Mutants to Dissect the Wheat Flowering Pathway

To establish a public reverse genetic tool to characterize the function of wheat genes, we developed a TILLING population of 2304 mutant lines from the tetraploid wheat cultivar Kronos. This population has a high mutation density (1/51kb) and yields on average 45 mutations per kb. To illustrate the value of this resource in the dissection of complex regulatory pathways, we characterized the effect of knockout mutants of the wheat vernalization gene VRN1. These vrn1 null-mutants exhibited very late flowering, but eventually produced fertile flowers and normal seeds. In both vrn1-null mutants and wild-type plants, the flowering repressor VRN2 was down-regulated during vernalization. After vernalization, however, VRN2 transcripts remained at very low levels in the wild-type plants but returned to high pre-vernalization levels in the vrn1-null mutants. These results demonstrate that a central role of VRN1 is to maintain low transcript levels of VRN2 in the leaves after the winter. The up-regulation of VRN2 delayed flowering by repressing the transcription of FT, a flowering-integrator gene that encodes a mobile protein that is transported from the leaves to the shoot apical meristem to induce flowering. The role of VRN2 in the delayed flowering of the vrn1-null mutants was confirmed by using a double vrn1-vrn2-null mutant, which flowered two months earlier than the vrn1-null mutants and also produced normal flowers and seeds. These results demonstrate that VRN1 is not essential for wheat flowering and that there are redundant flowering genes with similar functions to VRN1 (possibly the VRN1 paralogues FUL2 and/or FUL3).