A Novel Retrotransposon Inserted in the Dominant Vrn-B1 Allele Confers Spring Growth Habit in Tetraploid Wheat (Triticum turgidum L.)

Abstract

Vernalization genes determine winter/spring growth habit in temperate cereals and play important roles in plant development and environmental adaptation. In wheat (Triticum L. sp.), it was previously shown that allelic variation in the vernalization gene VRN1 was due to deletions or insertions either in the promoter or in the first intron. Here, we report a novel Vrn-B1 allele that has a retrotransposon in its promoter conferring spring growth habit. The VRN-B1 gene was mapped in a doubled haploid population that segregated for winter-spring growth habit but was derived from two spring tetraploid wheat genotypes, the durum wheat (T. turgidum subsp. durum) variety 'Lebsock' and T. turgidum subsp. carthlicum accession PI 94749. Genetic analysis revealed that Lebsock carried the dominant Vrn-A1 and recessive vrn-B1 alleles, whereas PI 94749 had the recessive vrn-A1 and dominant Vrn-B1 alleles. The Vrn-A1 allele in Lebsock was the same as the Vrn-A1c allele previously reported in hexaploid wheat. No differences existed between the vrn-B1 and Vrn-B1 alleles, except that a 5463-bp insertion was detected in the 5'-UTR region of the Vrn-B1 allele. This insertion was a novel retrotransposon (designated as retrotrans_VRN), which was flanked by a 5-bp target site duplication and contained primer binding site and polypurine tract motifs, a 325-bp long terminal repeat, and an open reading frame encoding 1231 amino acids. The insertion of retrotrans_VRN resulted in expression of Vrn-B1 without vernalization. Retrotrans_VRN is prevalent among T. turgidum subsp. carthlicum accessions, less prevalent among T. turgidum subsp. dicoccum accessions, and rarely found in other tetraploid wheat subspecies.

Keywords: Triticum turgidum; Vrn-B1; retrotransposon; tetraploid wheat; vernalization.

Figure 1

QTL mapping for growth habit segregation in the Lebsock × PI 94749–derived DH population. QTL analysis was performed with composite interval mapping using the computer program Map Manager QTX (Manly et al. 2001). The positions of marker loci are shown to the left of each linkage group and centiMorgan (cM) distances between loci are shown along the right. The vertical dotted line indicates the logarithm of the odds (LOD) significance threshold of 2.91. R² and LOD values are 0.30 and 19.67 for the QTL on chromosome 5AL (left), and 0.30 and 19.45 for the QTL on chromosome 5BL (right), respectively.

Figure 2

Markers for allelic variation in the first intron of VRN-A1. PCR products were amplified by primer pairs Ex1/C/F and Intr1/A/R3 (A) and Intr1/C/F and Intr1/AB/R (B) for detecting presence and absence of the large deletion in the first intron of VRN-A1, respectively. Lanes: M = size standard; L = Lebsock; P = PI 94749; VA and va are DH lines carrying dominant and recessive VRN-A1, respectively. Arrows indicate the expected size of the products.

Figure 3

Markers for allelic variation in VRN-B1. PCR products were amplified by primer pairs VRNBPF1 and VRNBPR1 (A), VRNBPF3 and VRNBPR1 (B), and VRNBPF1 and VBINSR1 (C). Lanes: M = size standard; L = Lebsock; P = PI 94749; VB and vb are DH lines carrying dominant and recessive VRN-B1, respectively. Arrows indicate the expected polymorphic PCR products.

Figure 4

Position and structure of retrotrans_VRN identified in the dominant Vrn-B1 allele of PI 94749. The aligned sequences were from positions at VRN-box to ATG start codon in PI 94749 (PI) and Lebsock (LB). The VRN-box and CArG-box are framed and shown in blue and pink, respectively. The ATG start codon is underlined. The duplicated target site CTCCG is shown in red with the position of retrotrans_VRN indicated by a red triangle. Positions of the primer binding site (PBS) and polypurine tract (PPT), long terminal repeat (LTR), and the coding region are all indicated. The numbers “−202” and “−101” indicate the start base positions in each line relative to start codon, and “−100” indicates the end base position in each line relative to the start codon. The DNA sequence of the 5.5-kb insertion in the dominant Vrn-B1 allele in PI 94749 is deposited in the EMBL/GenBank Data Libraries under accession no. HQ186251.

Figure 5

Expression of VRN-1 in DH lines derived from the cross between Lebsock and PI 94749. (Upper gel) Image of HinfI-digested cDNA fragments amplified by primers Ex4-5_F1 and Ex8_R1 for separating transcripts of VRN-A1 (313 bp) (indicated by long arrow) and the VRN-B1 (173 bp + 110 bp + 30 bp) (indicated by short arrows). (Lower gel) Actin was expressed at the same level in each of the lines carrying different VRN-1 alleles. Lanes: M = size standard; L = Lebsock; P = PI 94749; vernalized and unvernalized indicates plants with or without cold treatment at 4–6° for six weeks, respectively. VAVB, VAvb, vaVB, and vavb indicate DH lines having genotype of Vrn-A1Vrn-A1Vrn-B1Vrn-B1, Vrn-A1Vrn-A1vrn-B1vrn-B1, vrn-A1vrn-A1Vrn-B1Vrn-B1, and vrn-A1vrn-A1vrn-B1vrn-B1, accordingly. The third and ninth mean leaf samples were taken at the third and ninth leaf stages, respectively.