Molecular characterization of vernalization loci VRN1 in wild and cultivated wheats

Abstract

Background: Variability of the VRN1 promoter region of the unique collection of spring polyploid and wild diploid wheat species together with diploid goatgrasses (donor of B and D genomes of polyploid wheats) were investigated. Accessions of wild diploid (T. boeoticum, T. urartu) and tetraploid (T. araraticum, T. timopheevii) species were studied for the first time.

Results: Sequence analysis indicated great variability in the region from -62 to -221 nucleotide positions of the VRN1 promoter region. Different indels were found within this region in spring wheats. It was shown that VRN1 promoter region of B and G genome can also contain damages such as the insertion of the transposable element.Some transcription factor recognition sites including hybrid C/G-box for TaFDL2 protein known as the VRN1 gene upregulator were predicted inside the variable region. It was shown that deletions leading to promoter damage occurred in diploid and polyploid species independently. DNA transposon insertions first occurred in polyploid species. At the same time, the duplication of the promoter region was observed in A genomes of polyploid species.

Conclusions: We can conclude that supposed molecular mechanism of the VRN1 gene activating in cultivated diploid wheat species T. monococcum is common also for wild T. boeoticum and was inherited by T. monococcum. The spring polyploids are not related in their origin to spring diploids. The spring T. urartu and goatgrass accessions have another mechanism of flowering activation that is not connected with indels in VRN1 promoter region. All obtained data may be useful for detailed insight into origin of spring wheat forms in evolution and domestication process.

Figure 1

PCR amplification of VRN1 promoter region with primers AP1_ProDel_F1/AP1_ProDel_R1. A. Diploid wheat accessions. T_b - T. boeoticum, T_m - T. monococcum, Ae_sp - Ae. speltoides, Ae_sq - Ae. squarrosa. T_b PI-427328, K-20741, K-40118 have 20 bp deletion. W - winter growth habit. B. Spring diploid Triticum and Aegilops accessions. T_b IG-45296 has 20 bp deletion, T_m K-18105 has 34 bp deletion. M - Marker-Bioline HyperLader IV.

Figure 2

Deletions observed in the VRN1 promoter alleles among spring diploid species. Nucleotide positions are indicated according to T. aestivum vrn-A1 (GenBank: AY616455) promoter. In boxes there were represented 3 letter repeats flanked the place of the deletions. The AP1_ProDel_R1 primer annealing site is indicated by solid line and 6 bp repeat near this region in A genome - by dashed line. T_m -T. monococcum, T_u - T. urartu, T_b - T. boeoticum, T_ae - T. aestivum, Ae_sp - Ae. speltoides, Ae_sq - Ae. squarrosa. T_ae_TDC A- vrn-A1 (AY616455), T_ae_TDC B- vrn-B1 (AY616456), T_ae_TDC D- vrn-D1 (AY616457) were obtained from GenBank.

Figure 3

Indels and regulatory sites found in VRN1 promoter region. A. Schematic representation of the different VRN1 promoter variants observed in spring wheat accessions. Places of indels are marked in bp. numbers upstream from the start codon. Grey boxes indicate deletions and black triangles - insertions. One of the direct repeats flanked deletions that remain in sequence after recombination is outside the grey box. B. The predicted transcription factors binding sites.

Figure 4

Alignment of foldback elements insertion observed in different VRN1 alleles. T_ae-T. aestivum, T_ti-T. timopheevii, T_tu-T. turgidum, TDD-T. aestivum (near-isogenic line Triple Dirk D).

Figure 5

Variable VRN-A1 promoter copies. Boxes depict deletions (small black box -8 bp deletion), triangles - transposon insertion. T_m -T. monococcum, T_u - T. urartu , T_b - T. boeoticum, T_tu - T. turgidum, T_di - T. dicoccum, T_du - T. durum, T_t - T. turanicum, T_ti - T. timopheevii, T_ar - T. araraticum, T_ae Sar - T. aestivum s: Saratovskaya/Vietnamskaya 5R(5A), T_ae Pir - T. aestivum cv. Pyrothrix 28, T_ae Mir - T. aestivum cv. Mironovskaya yarovaya, T_ae Jup - T. aestivum cv. Jupateko. T_b/T_m 1,2 and T_t 1,2 show two different allele's variants between accessions of indicated species. Accession number: * - T. m. winter -PI94743, spring - K-20400, Mute KT3-5, K-20970, PI306540; T. b. winter - G1777, K-25811, spring - K-14384, PI428217, IG116196. ** - T. m. spring - K-18105 (Vrn-A1g); T. b. spring - IG116198, PI427328, K-20741, IG45296, K-40118, KU8136, KU8120, KU8059 (Vrn-A1h), K-40117 (Vrn-A1g).