Copy number and haplotype variation at the VRN-A1 and central FR-A2 loci are associated with frost tolerance in hexaploid wheat

Abstract

The interaction between VRN - A1 and FR - A2 largely affect the frost tolerance of hexaploid wheat. Frost tolerance is critical for wheat survival during cold winters. Natural variation for this trait is mainly associated with allelic differences at the VERNALIZATION 1 (VRN1) and FROST RESISTANCE 2 (FR2) loci. VRN1 regulates the transition between vegetative and reproductive stages and FR2, a locus including several tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors, regulates the expression of Cold-regulated genes. We identified sequence and copy number variation at these two loci among winter and spring wheat varieties and characterized their association with frost tolerance. We identified two FR-A2 haplotypes-'FR-A2-S' and 'FR-A2-T'-distinguished by two insertion/deletions and ten single nucleotide polymorphisms within the CBF-A12 and CBF-A15 genes. Increased copy number of CBF-A14 was frequently associated with the FR-A2-T haplotype and with higher CBF14 transcript levels in response to cold. Factorial ANOVAs revealed significant interactions between VRN1 and FR-A2 for frost tolerance in both winter and spring panels suggesting a crosstalk between vernalization and cold acclimation pathways. The model including these two loci and their interaction explained 32.0 and 20.7 % of the variation in frost tolerance in the winter and spring panels, respectively. The interaction was validated in a winter wheat F 4:5 population segregating for both genes. Increased VRN-A1 copy number was associated with improved frost tolerance among varieties carrying the FR-A2-T allele but not among those carrying the FR-A2-S allele. These results suggest that selection of varieties carrying the FR-A2-T allele and three copies of the recessive vrn-A1 allele would be a good strategy to improve frost tolerance in wheat.

Fig. 1

Nucleotide and amino acid sequence variation distinguishing FR-A2 haplotypes Nucleotide positions of the two indel and ten SNP polymorphisms of CBF-A12 and CBF-A15 are listed. Six of the 12 sequence polymorphisms lead to amino acid changes and the positions and detail of each amino acid substitution in CBF-12 and CBF-15 are displayed. AP2, CMIII-1, CMIII-2, CMIII-3 and CMIII-4 are the five conserved amino acid motifs which distinguish CBF genes from the other members of ERF family. These five domains are indicated by different colors and filling types in the diagrammatic sketch of CBF12 and CBF15 proteins

Fig. 2

Interaction between VRN-A1 and FR-A2 within the winter panel (a), Eltan/ORFW F_4:5 population (b) and the spring panel (c). For each class and allele combination, average LS means survival rates ± SE are presented. All the interactions were highly significant (P<0.05, based on ANOVA including VRN-A1 and FR-A2 for each population, Table 5).

Fig. 3

Expression levels of CBF12, CBF14 and CBF15 at 25 °C (a – c) and after 8 h exposure to 4 °C (d – f). Y-axis values represent the ratio between the initial number of molecules of the target gene and the number of molecules of ACTIN and are the average of six biological replicates ± SE. V/W = Vrn-A1 allele, S = ‘FR-A2-S’, T = ‘FR-A2-T’. * = P ≤ 0.05, *** = P ≤ 0.001, significance of differences between ‘FR-A2-S’ and ‘FR-A2-T’.