Development and characterization of a spring hexaploid wheat line with no functional VRN2 genes

Abstract

The combination of three non-functional alleles of the flowering repressor VRN2 results in a spring growth habit in wheat. In temperate cereals with a winter growth habit, a prolonged exposure to low temperatures (vernalization) accelerates flowering. Before vernalization, the VRN2 locus plays a central role in maintaining flowering repression. Non-functional VRN2 alleles result in spring growth habit and are frequent in diploid wheat and barley. However, in hexaploid wheat, the effect of these non-functional VRN2 alleles is masked by gene redundancy. In this study, we developed a triple VRN2 mutant (synthetic vrn2-null) in hexaploid wheat by combining the non-functional VRN-A2 allele present in most polyploid wheats with a VRN-B2 deletion from tetraploid wheat, and a non-functional VRN-D2 allele from Aegilops tauschii (Ae. tauschii) (the donor of hexaploid wheat D genome). Non-vernalized vrn2-null plants flowered 118 days (P < 2.8E-07) earlier than the winter control, and showed a limited vernalization response. The functional VRN-B2 allele is expressed at higher levels than the functional VRN-D2 allele and showed a stronger repressive effect under partial vernalization (4 °C for 4 weeks), and also in non-vernalized plants carrying only a functional VRN-B2 or VRN-D2 in heterozygous state. These results suggest that different combinations of VRN-B2 and VRN-D2 alleles can be a used to modulate the vernalization response in regions with mild winters. Spring vrn2-null mutants have been selected repeatedly in diploid wheat and barley, suggesting that they may have an adaptative value and that may be useful in hexaploid wheat. Spring wheat breeders can use these new alleles to improve wheat adaptation to different or changing environments.

Fig. 1

Effect of VRN2 mutations on flowering time in diploid Ae. tauschii. Heading time of 71 F₂ plants from the cross between Ae. tauschii accessions E1 (early flowering) and AS75 (late flowering). A = AS75 allele, B = Ae. tauschii E1 allele and H = heterozygous. E1 = Ae. tauschii E1 and AS75 = Ae. tauschii AS75 indicate parental controls. Plants were grown under long days in the absence of vernalization. ***P < 0.001

Fig. 2

Mutations in the VRN 2 locus of Ae. tauschii E1. Multiple alignments of predicted coding sequences of ZCCT1 a and ZCCT2 b in Ae. tauchii E1 (KM489155 and KM489156), and winter controls AS75 (KM503042 and KM503043) and AL8/78 (ACI00354 and ACI00358). Numbers in parenthesis are GenBank accession numbers for ZCCT1 and ZCCT2, respectively. For the ZCCT1 protein from E1, a 35 amino acid deletion observed in 87.5 % of the cloned and sequenced cDNAs is underlined (the other 12.5 % of the clones include an intron region producing a premature stop codon). The CCT domain is indicated with asterisks in both genes. SNPs are highlighted in gray. Critical Arg residues in the CCT domain are highlighted in black

Fig. 3

Development of synthetic vrn2-null hexaploid wheat. a Introgression of vrn-A2 and vrn-B2 in the tetraploid cultivar Kronos. b Production of synthetic aneuploid to introgress vrn-D2, self-pollination to recover chromosome number. c Backcross with winter hexaploid wheat to produce a segregating line for vrn-A2, vrn-B2 and vrn-D2 in a winter background. X represents a crossing step, arrow represents a progeny. Non-functional VRN2 alleles are underlined

Fig. 4

Effect of mutations in VRN-B2 and VRN-D2 on heading time under non vernalizing conditions. a Heading times for the triple mutant. Bars represent mean and error bars represent the SEM. Dashes represent mutant alleles, wt: wild type alleles and H: heterozygous. Lower doses of functional VRN2 were enough to significantly delay flowering. **P < 0.005 ***P < 0.001. Arrow indicates that the experiment was stopped before heading. b Maturity differences between the triple VRN2 mutant (left) and the wild type (right)

Fig. 5

Effects of mutations in VRN-B2 and VRN-D2 on heading time under different vernalization treatments. Error bars represent the SE of the mean of 5 biological replications. Dashes represent a non-functional allele and wt a wild type allele. For each treatment different letters indicate significant differences (Tukey’s test, P < 0.05)

Fig. 6

Transcript levels of ZCCT-B2 and ZCCT-D2 in hexaploid wheat. ZCCT-B2 levels are 6 and 4 times higher than ZCCT-D2 in TDC and vrn2-synthetic, respectively. Triple Dirk C (TDC) is a winter hexaploid line and vrn2-synthetic corresponds to the wild type sister line of the synthetic vrn2-null developed in this study. Bars represent means of 5 biological and 2 technical replications. Plants were grown under long day conditions (16 h light) at a temperature of 20 °C day/18 °C night for 3 weeks. Samples were collected at ZT 4 (Zeitgeber Time). *P < 0.001