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

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.

SUBMITTER: Devisetty UK

PROVIDER: S-EPMC2962619 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Devisetty Upendra Kumar UK Mayes Katie K Mayes Sean S

BMC research notes 20100929

<h4>Background</h4>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 comp ...[more]

PMID: 20920212

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Project description:BACKGROUND:Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. RESULTS:We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. CONCLUSIONS:The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

Project description:The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress.Sequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains.The high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.

Project description:BackgroundThe patterns of expression of homoeologous genes in hexaploid bread wheat have been intensively studied in recent years, but the interaction between structural genes and their homoeologous regulatory genes remained unclear. The question was as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The aim of the present study was cloning, sequence analysis, mapping and expression analysis of F3H (flavanone 3-hydroxylase - one of the key enzymes in the plant flavonoid biosynthesis pathway) homoeologues in bread wheat and study of the interaction between F3H and their regulatory genes homoeologues - Rc (red coleoptiles).ResultsPCR-based cloning of F3H sequences from hexaploid bread wheat (Triticum aestivum L.), a wild tetraploid wheat (T. timopheevii) and their putative diploid progenitors was employed to localize, physically map and analyse the expression of four distinct bread wheat F3H copies. Three of these form a homoeologous set, mapping to the chromosomes of homoeologous group 2; they are highly similar to one another at the structural and functional levels. However, the fourth copy is less homologous, and was not expressed in anthocyanin pigmented coleoptiles. The presence of dominant alleles at the Rc-1 homoeologous loci, which are responsible for anthocyanin pigmentation in the coleoptile, was correlated with F3H expression in pigmented coleoptiles. Each dominant Rc-1 allele affected the expression of the three F3H homoeologues equally, but the level of F3H expression was dependent on the identity of the dominant Rc-1 allele present. Thus, the homoeologous Rc-1 genes contribute more to functional divergence than do the structural F3H genes.ConclusionThe lack of any genome-specific relationship between F3H-1 and Rc-1 implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat. Regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.

Project description:5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized.Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring "Louise'. Development of genome-specific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis.The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7 % identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100 % identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7 % identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice).The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance.

Dataset Information

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

Publications

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

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