The occurrence of spring forms in tetraploid Timopheevi wheat is associated with variation in the first intron of the VRN-A1 gene

Abstract

Background: Triticum araraticum and Triticum timopheevii are tetraploid species of the Timopheevi group. The former includes both winter and spring forms with a predominance of winter forms, whereas T. timopheevii is considered a spring species. In order to clarify the origin of the spring growth habit in T. timopheevii, allelic variability of the VRN-1 gene was investigated in a set of accessions of both tetraploid species, together with the diploid species Ae. speltoides, presumed donor of the G genome to these tetraploids.

Results: The promoter region of the VRN-A1 locus in all studied tetraploid accessions of both T. araraticum and T. timopheevii represents the previously described allele VRN-A1f with a 50 bp deletion near the start codon. Three additional alleles were identified namely, VRN-A1f-del, VRN-A1f-ins and VRN-A1f-del/ins, which contained large mutations in the first (1^st) intron of VRN-A1. The first allele, carrying a deletion of 2.7 kb in a central part of intron 1, occurred in a few accessions of T. araraticum and no accessions of T. timopheevii. The VRN-A1f-ins allele, containing the insertion of a 0.4 kb MITE element about 0.4 kb upstream from the start of intron 1, and allele VRN-A1f-del/ins having this insertion coupled with a deletion of 2.7 kb are characteristic only for T. timopheevii. Allelic variation at the VRN-G1 locus includes the previously described allele VRN-G1a (with the insertion of a 0.2 kb MITE in the promoter) found in a few accessions of both tetraploid species. We showed that alleles VRN-A1f-del and VRN-G1a have no association with the spring growth habit, while in all accessions of T. timopheevii this habit was associated with the dominant VRN-A1f-ins and VRN-A1f-del/ins alleles. None of the Ae. speltoides accessions included in this study had changes in the promoter or 1^st intron regions of VRN-1 which might confer a spring growth habit. The VRN-1 promoter sequences analyzed herein and downloaded from databases have been used to construct a phylogram to assess the time of divergence of Ae. speltoides in relation to other wheat species.

Conclusions: Among accessions of T. araraticum, the preferentially winter predecessor of T. timopheevii, two large mutations were found in both VRN-A1 and VRN-G1 loci (VRN-A1f-del and VRN-G1a) that were found to have no effect on vernalization requirements. Spring tetraploid T. timopheevii had one VRN-1 allele in common for two species (VRN-G1a), and two that were specific (VRN-A1f-ins, VRN-A1f-del/ins). The latter alleles include mutations in the 1^st intron of VRN-A1 and also share a 0.4 kb MITE insertion near the start of intron 1. We suggested that this insertion resulted in a spring growth habit in a progenitor of T. timopheevii which has probably been selected during subsequent domestication. The phylogram constructed on the basis of the VRN-1 promoter sequences confirmed the early divergence (~3.5 MYA) of the ancestor(s) of the B/G genomes from Ae. speltoides.

Keywords: Aegilops; Allelic variation; First intron; Promoter; Triticum; VRN-1 gene; Vernalization.

Fig. 1

Schematic representation of different VRN-1 alleles including promoter, 1^st exon (grey rectangle) and 1^st intron. The positions of specific primers are shown above each scheme. Deletions and insertions of MITE transposable elements are indicated by empty and filled triangles, respectively, with sizes (bp) above

Fig. 2

PCR amplification with primers VrnA1F/Int1R (a), P1/P5 (b), Intr1/C/F // Intr1/AB/R (c), delf/delr (d), mitef/miter (e), Ex1/C/F // Intr1/B/R4 (f). Accession numbers, species and genotype are given at the top

Fig. 3

Schemes of different VRN-1 alleles with insertions of MITE elements indicated. The putative TATA- box (TTAAAAA) and CArG-box are depicted. The exact position of indels and regulatory sites within the promoter region is marked by the number of bases counted from the start codon

Fig. 4

The neighbor-joining phylogram of species based on the alignment of the nucleotide VRN-1 promoter sequences. Letters in bold within genotypes indicate genomes from which the corresponding sequences were isolated. The numbers above and below forks indicate bootstrap values and times of divergence, respectively. Scale of divergence time in MYA is shown below the phylogram. Sequences were selected covering, at least, 0.8 kb of the promoter region. The sequences analysed herein are marked with an asterisk