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. 2010 Sep 29:3:245.
doi: 10.1186/1756-0500-3-245.

The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis

Upendra Kumar Devisetty  1 , Katie Mayes, Sean Mayes
Affiliations

The RAD51 and DMC1 homoeologous genes of bread wheat: cloning, molecular characterization and expression analysis

Upendra Kumar Devisetty et al. BMC Res Notes. .

Abstract

Background: Meiotic recombination in eukaryotes requires two homologues of the E. coli RecA proteins: Rad51 and Dmc1. Both proteins play important roles in the binding of single stranded DNA, homology search, strand invasion and strand exchange. Meiotic recombination has been well studied in Arabidopsis, rice, maize and the orthologues of RAD51 and DMC1 have been characterized. However genetic analysis of the RAD51 and DMC1 genes in bread wheat has been hampered due to the absence of complete sequence information and because of the existence of multiple copies of each gene in the hexaploid wheat genome.

Findings: In this study we have identified that TaRAD51 and TaDMC1 homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the TaRAD51 and TaDMC1 homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of TaRAD51 and TaDMC1 and those of other eukaryotes reveal a high degree of evolutionary conservation. Despite the high degree of sequence conservation at the nucleotide level, genome-specific primers for cDNAs of TaRAD51 and TaDMC1 were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the TaRAD51 and TaDMC1 cDNA homoeologues was largely restricted to meiotic tissue, with elevated levels observed during the stages of prophase I when meiotic recombination occurs. All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.

Conclusions: Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues. While differences were detected between the three cDNA homoeologues of TaRAD51 as well as the three homoeologues of TaDMC1, it is unlikely that the predicted amino acid substitutions would have an effect on the protein structure, based on our three-dimensional structure prediction analyses. There are differences in the levels of expression of the three homoeologues of TaRAD51 and TaDMC1 as determined by QRT-PCR and if these differences are reflected at the protein level, bread wheat may be more dependent upon a particular homoeologue to achieve full fertility than all three equally.

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Figures

Figure 1
Figure 1
PCR assay for the chromosomal localization of the TaRAD51 and TaDMC1 homoeologous loci, respectively, on group 7 and group 5 of hexaploid wheat. Each primer set designed for TaRAD51 was used to amplify NT7A7B, NT7A7D, NT7B7A, NT7B7D, NT7D7A, NT7D7B, water control and CS. (a) TaRAD51 (A) and (D) genome-specific primer PCR amplifications. The two bands (indicated by red arrows) in Nullisomic 7B and tetrasomic for 7A and 7D lanes can be allocated either to A or D genomes based on PCR amplification on other Nullisomics (Nullisomics 7A and 7D); TaRAD51(B) genome-specific primer PCR amplification and absence of bands in the line nullisomic for 7B and tetrasomic for both 7A and 7D allocates this primer to genome 7B. Each primer set for TaDMC1 was used to amplify NT5A5B, NT5B5A, NT5D5A, water control and CS (b) TaDMC1(A), (B) & (D) genome-specific primer PCR amplification. Absence of bands in the group 5 Nullisomics allocates the primer to the respective genomes; M = 2-log ladder (NEB). Only group 5 and group 7 of Nulli-tetrasomics are shown.
Figure 2
Figure 2
Alignment of 3'- and 5'-UTR sequences of TaRAD51 and TaDMC1 homoeologues from hexaploid wheat indicating the location of genome specific primers. (a) TaRAD51 cDNA genome-specific primer design. (b) TaDMC1 cDNA genome-specific primer design. Deletions are shown by dashes. The positions of the 3' and 5' UTRs in relation to the start codon 'A' are indicated at the top of the sequence. The sequence of the forward primer and complementary sequences of the reverse primers are shown in bold. The black arrows indicate the position of forward and reverse primers.
Figure 3
Figure 3
PCR assay for genome specificity of TaRAD51 and TaDMC1 cDNA homoeologues in bread wheat. Each primer set for TaRAD51 was used to amplify NT7A7B, NT7A7D, NT7B7A, NT7B7D, NT7D7A, NT7D7B, water control and CS (a) TaRAD51(A), (B) & (D) genome-specific primer PCR amplification. Each primer set for TaDMC1 was used to amplify NT5A5B, NT5A5D, NT5B5A, NT5B5D, NT5D5A, NT5D5B, water control and CS.(b) TaDMC1(A), (B) & (D) genome-specific primer PCR amplification. Absence of bands in the group 7 & 5 Nullisomics allocates the primer to the respective genomes; M = 2-log ladder (NEB).
Figure 4
Figure 4
The deduced amino acids alignments of TaRAD51 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaRAD51 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three cDNA homoeologous proteins are indicated by black with a grey background. (b) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaRAD51. (c) The 3D structure of TaRad51-7A is represented in red, TaRad51-7B in green and TaRad51-7D in yellow. Blue arrow shows the magnified image of the side chains of three TaRad51 homoeologue proteins, which indicates the only structural dissimilarity.
Figure 5
Figure 5
The deduced amino acids alignments of TaDMC1 homoeologous proteins and their 3D models. (a) Multiple alignments of the three TaDMC1 sequences identified in the three bread wheat genomes (A, B and D). Conserved amino acids are indicated by black with a yellow background. The amino acid differences between the three homoeologous proteins are indicated by black with a grey background. (B) SIFT predictions for the amino acid substitutions for the three cDNA homoeologues of TaDMC1. (C) The predicted 3D structure of TaDmc1-7A is represented in red, TaDmc1-7B in green and TaDmc1-7D in yellow.
Figure 6
Figure 6
Evolutionary tree of Rad51 and Dmc1 proteins. Phylogenetic tree obtained from a nucleotide alignment of cDNAs derived from the TaRAD51 and TaDMC1 homoeologues together with a wide range of RAD51 and DMC1 orthologues using the Maximum Likelihood method.
Figure 7
Figure 7
QRT-PCR expression analysis of the TaRAD51 and TaDMC1 homoeologous genes. (a) The green bars represents TaRAD51(A), pink bar represents TaRAD51(B) and blue bar represents TaRAD51(D). (B) The green bars represents TaDMC1(A), pink bar represents TaDMC1(B) and blue bar represents TaDMC1(D). Abbreviations used: PM, pre-meiotic interphase; LP, leptotene-pachytene, DA, diplotene-anaphase I; TT, telophase I-telophase II; T, tetrad; IP, immature pollen; RT, root tips; L, leaves
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