Positional cloning of the wheat vernalization gene VRN1

Abstract

Winter wheats require several weeks at low temperature to flower. This process, vernalization, is controlled mainly by the VRN1 gene. Using 6,190 gametes, we found VRN1 to be completely linked to MADS-box genes AP1 and AGLG1 in a 0.03-centimorgan interval flanked by genes Cysteine and Cytochrome B5. No additional genes were found between the last two genes in the 324-kb Triticum monococcum sequence or in the colinear regions in rice and sorghum. Wheat AP1 and AGLG1 genes were similar to Arabidopsis meristem identity genes AP1 and AGL2, respectively. AP1 transcription was regulated by vernalization in both apices and leaves, and the progressive increase of AP1 transcription was consistent with the progressive effect of vernalization on flowering time. Vernalization was required for AP1 transcription in apices and leaves in winter wheat but not in spring wheat. AGLG1 transcripts were detected during spike differentiation but not in vernalized apices or leaves, suggesting that AP1 acts upstream of AGLG1. No differences were detected between genotypes with different VRN1 alleles in the AP1 and AGLG1 coding regions, but three independent deletions were found in the promoter region of AP1. These results suggest that AP1 is a better candidate for VRN1 than AGLG1. The epistatic interactions between vernalization genes VRN1 and VRN2 suggested a model in which VRN2 would repress directly or indirectly the expression of AP1. A mutation in the promoter region of AP1 would result in the lack of recognition of the repressor and in a dominant spring growth habit.

Figure 1

(A) Genetic map of the VRN1 region on chromosome 5A^m of T. monococcum. Genetic distances are in cM (6,190 gametes). (B–D) Physical maps of the colinear VRN1 regions in rice, sorghum, and wheat. Regions indicated in red have been sequenced. Double dot lines indicate gaps in the current physical maps. (B) Sequence of the colinear region in rice chromosome 3. (C) S. bicolor BACs 170F8 (AF503433) and 17E12 (AY188330). (D) T. monococcum physical map. BAC clones order from left to right is: 49I16, 115G1, 136F13, 133P9, 116F2, 89E14, 160C18, 491M20, 328O3, 609E6, 393O11, 719C13, 454P4, 54K21, 579P2, 601A24, 231A16, 638J12, 52F19, 242A12, 668L22, 539M19, and 309P20 (bold indicates sequenced BACs). Black dots indicate validation of BAC connections by hybridization. (E) Gene structure of two MADS-box genes completely linked to the VRN1 gene (AY188331, AY188333). Bars represent exons. (F) Sequence comparison of the AP1 promoter regions from genotypes carrying the Vrn1 and vrn1 alleles and from two T. monococcum accessions with additional deletions. The last two accessions are spring but their genotype has not been determined yet. Numbers indicate distances from the start codon. A putative MADS-box protein-binding site (CArG-box) is indicated in green.

Figure 2

(A) RT-PCR experiment using T. monococcum G3116 (winter growth habit) and AP1-, AGLG1-, and actin-specific primers. The PCRs for the three genes were performed by using the same cDNA samples. Lanes 1–5 indicate leaves. Lane 1, before vernalization; lanes 2–4, 2, 4, and 6 weeks of vernalization, respectively; lane 5, 2 weeks after vernalized plants were returned to the greenhouse; lane 6, unvernalized apices; lane 7, 6-week vernalized apices; and lane 8, young spikes. (B) AP1 transcription levels in leaves relative to actin measured by quantitative PCR. Lanes 1–5, leaves from plants at the same vernalization stage as samples 1–5 in A. Units are values linearized by using the ) method, where C_T is the threshold cycle.

Figure 3

A model of the regulation of flowering initiation by vernalization in wheat.