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

Devisetty UK, Mayes K, Mayes S - BMC Res Notes (2010)

Bottom Line: All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Crop sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, UK. sean.mayes@nottingham.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus

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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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

Mentions: The QRT-PCR analysis of TaRAD51 cDNA homoeologues indicated that all the three homoeologues were expressed in both vegetative and meiotic tissues (Figure 7a). Comparatively higher expression levels were observed in meiotic stages than vegetative tissues (Figure 7a, stages PM to TT). There is a sharp drop in expression of the TaRAD51 cDNA homoeologue transcripts at the end of Meiosis I but then expression increases again at the Tetrad stage. There was however differences in expression level among the three cDNA homoeologues, particularly during meiotic stages. This may suggest that the individual homoeologues contribute to differing extents in meiotic recombination. The expression level of TaRAD51-7B was significantly higher than TaRAD51-7A and TaRAD51-7D in all meiotic stages suggesting it could be the version with the greatest role in meiosis.


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

Devisetty UK, Mayes K, Mayes S - BMC Res Notes (2010)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962619&req=5

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
Mentions: The QRT-PCR analysis of TaRAD51 cDNA homoeologues indicated that all the three homoeologues were expressed in both vegetative and meiotic tissues (Figure 7a). Comparatively higher expression levels were observed in meiotic stages than vegetative tissues (Figure 7a, stages PM to TT). There is a sharp drop in expression of the TaRAD51 cDNA homoeologue transcripts at the end of Meiosis I but then expression increases again at the Tetrad stage. There was however differences in expression level among the three cDNA homoeologues, particularly during meiotic stages. This may suggest that the individual homoeologues contribute to differing extents in meiotic recombination. The expression level of TaRAD51-7B was significantly higher than TaRAD51-7A and TaRAD51-7D in all meiotic stages suggesting it could be the version with the greatest role in meiosis.

Bottom Line: All three homoeologues of both strand-exchange proteins (TaRAD51 and TaDMC1) are expressed in wheat.Bread wheat contains three expressed copies of each of the TaRAD51 and TaDMC1 homoeologues.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Crop sciences, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, UK. sean.mayes@nottingham.ac.uk.

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.

No MeSH data available.


Related in: MedlinePlus