Molecular investigation of RAD51 and DMC1 homoeologous genes of hexaploid wheat (Triticum aetivum L.)

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis

Molecular investigation of RAD51 and DMC1 homoeologous genes of hexaploid wheat (Triticum aetivum L.)

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis

Polymorphism identification and improved genome annotation of Brassica rapa through Deep RNA sequencing.

The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. This is useful for accurate mRNA abundance and detection of expression QTL (eQTLs) in mapping populations. Deep RNA-Seq of two Brassica rapa genotypes-R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)-using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. A total of 330,995 SNPs were identified in transcribed regions between the two genotypes with an average frequency of one SNP in every 200 bases. The deep RNA-Seq reassembled Brassica rapa transcriptome identified 44,239 protein-coding genes. Compared with current gene models of B. rapa, we detected 3537 novel transcripts, 23,754 gene models had structural modifications, and 3655 annotated proteins changed. Gaps in the current genome assembly of B. rapa are highlighted by our identification of 780 unmapped transcripts. All the SNPs, annotations, and predicted transcripts can be viewed at http://phytonetworks.ucdavis.edu/

RNA-Seq analysis of soft rush (Juncus effusus): transcriptome sequencing, de novo assembly, annotation, and polymorphism identification

Background: Juncus effusus L. (family: Juncaceae; order: Poales) is a helophytic rush growing in temperate damp or wet terrestrial habitats and is of almost cosmopolitan distribution. The species has been studied intensively with respect to its interaction with co-occurring plants as well as microbes being involved in major biogeochemical cycles. J. effusus has biotechnological value as component of Constructed Wetlands where the plant has been employed in phytoremediation of contaminated water. Its genome has not been sequenced. Results: In this study we carried out functional annotation and polymorphism analysis of de novo assembled RNA-Seq data from 18 genotypes using 249 million paired-end Illumina HiSeq reads and 2.8 million 454 Titanium reads. The assembly comprised 158,591 contigs with a mean contig length of 780bp. The assembly was annotated using the dammit! annotation pipeline, which queries the databases OrthoDB, Pfam-A, Rfam, and runs BUSCO (Benchmarking Single-Copy Ortholog genes). In total, 111,567 contigs (70.3%) were annotated with functional descriptions, assigned gene ontology terms, and conserved protein domains, which resulted in 30,932 non-redundant gene sequences. Results of BUSCO and KEGG pathway analyses were similar for J. effusus as for the well-studied members of the Poales, Oryza sativa and Sorghum bicolor. A total of 566,433 polymorphisms were identified in transcribed regions with an average frequency of 1 polymorphism in every 171 bases. Conclusions: The transcriptome assembly was of high quality and genome coverage was sufficient for global analyses. This annotated knowledge resource can be utilized for future gene expression analysis, genomic feature comparisons, genotyping, primer design, and functional genomics in J. effusus.German Academic Exchange Program (DAAD); German Science Foundation (DFG) [MI 1500/2-1]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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

<p>Abstract</p> <p>Background</p> <p>Meiotic recombination in eukaryotes requires two homologues of the <it>E. coli </it>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 <it>RAD51 </it>and <it>DMC1 </it>have been characterized. However genetic analysis of the <it>RAD51 </it>and <it>DMC1 </it>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.</p> <p>Findings</p> <p>In this study we have identified that <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues are located on group 7 and group 5 chromosomes of hexaploid wheat, respectively. Comparative sequence analysis of cDNA derived from the <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues revealed limited sequence divergence at both the nucleotide and the amino acid level. Indeed, comparisons between the predicted amino acid sequences of <it>TaRAD51 </it>and <it>TaDMC1 </it>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 <it>TaRAD51 </it>and <it>TaDMC1 </it>were developed to evaluate expression patterns of individual homoeologues during meiosis. QRT-PCR analysis showed that expression of the <it>TaRAD51 </it>and <it>TaDMC1 </it>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 (<it>TaRAD51 </it>and <it>TaDMC1</it>) are expressed in wheat.</p> <p>Conclusions</p> <p>Bread wheat contains three expressed copies of each of the <it>TaRAD51 </it>and <it>TaDMC1 </it>homoeologues. While differences were detected between the three cDNA homoeologues of <it>TaRAD51 </it>as well as the three homoeologues of <it>TaDMC1</it>, 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 <it>TaRAD51 </it>and <it>TaDMC1 </it>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.</p

Molecular investigation of RAD51 and DMC1 homoeologous genes of hexaploid wheat (Triticum aetivum L.)

Meiotic recombination in eukaryotes requires two orthologues of the E. coli RecA proteins, Rad51 and Dmc1. Both genes play an important role in the binding of single strand DNA, homology search, strand invasion and strand exchange resulting in Holliday junctions which are resolved into crossovers or non-crossovers events. Even though both genes are well characterized in a variety of organisms including plants, very little information is available from hexaploid wheat. In most diploid plant species, deletion of either the RAD51 or DMC1 orthologues leads to sterility but wheat being a polyploid, offers a unique opportunity to examine the effects of the deletion of specific homoeologue, while maintaining a degree of fertility. The transcript expression profiling of RAD51 and DMC1 genes in Arabidopsis, rice and wheat using available microarray databases indicated higher levels of expression in mitotically and meiotically active tissues compared to other tissues. However, the possible function of the DMC1 gene in mitotic-active tissues needs to be investigated further. Previously cDNA sequences of TaRAD51 and TaDMCl of hexaploid wheat were cloned and reported. In this study, it has been demonstrated that the reported TaRAD51A1 and TaRAD51A2 cDNA sequences are (D) and (A) homoeologues of TaRAD51 respectively and TaDMCl cDNA sequence is (D) homoeologue of the TaDMC1. This study also found that the amino acid sequences and evolutionary relationships of RAD51 and DMC1 cDNA homoeologues are highly conserved across eukaryotes. Functional characterization of TaRAD51 and TaDMCl gene homoeologues was undertaken in planta using Forward Genetics, Reverse Genetics and Complementation methods. Forward and Reverse Genetic screening of a subset of a Highbury mutant population could not identify any mutants that have deletions in TaRAD51 and TaDMC1 genes. However, Reverse Genetics screening of Paragon mutant population identified mutant lines that tested as having deletions for all the three homoeologues of TaRAD51 and TaDMCl. However, most likely due to high mutational load and a deleterious phenotype, only a few mutant lines survived. Phenotypic and cytogenetic analysis indicated the probable functional redundancy of TaRAD51 (B) homoeologue in meiosis, although the unknown size of the deletion and limited phenotype makes it impossible to completely certain of this. The single mutants for TaDMC1 (B) and (D) indicated a reduction in pollen viability and ear fertility compared to wild-type. The cytological examination of these mutants indicated low levels of abnormal diakinesis, resulting in the formation of dyads. However, the single mutants were still able to produce normal tetrads. This suggests that there is a possible dosage effect of these homoeologues in hexaploid wheat. Unless deletion lines for the (A) and (D) homoeologues of TaRAD51 and (B) homoeologue of TaDMC1 can be recovered and characterized the above assumptions will remain inconclusive. The results of complementation assays using over-expressing CaMV35S::TaRAD51(D)±GFP constructs demonstrated a very low (-14% and -2%, respectively, with +GFP and -OFP constructs) functional complementation in terms of seed set compared to 0% in homozygous Atrad51 mutants. One explanation of these results is that the wheat genes are not complete functional orthologues for the inactivated Arabidopsis genes. The functional complementation experiments could not be performed for TaDMC1 gene because of time limitation, although the transformants were produced in AtDMC1/atdmc1 background. Finally, overexpression of the TaRAD51 gene suggests 2-fold increase in genetic distances in Arabidopsis using CaMV35S::TaRAD51(D) construct. This was done by crossing the appropriate transformant with fluorescent tetrad lines. However the results need to be confirmed by a large scale analysis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Bringing your tools to CyVerse Discovery Environment using Docker [version 3; referees: 3 approved]

Docker has become a very popular container-based virtualization platform for software distribution that has revolutionized the way in which scientific software and software dependencies (software stacks) can be packaged, distributed, and deployed. Docker makes the complex and time-consuming installation procedures needed for scientific software a one-time process. Because it enables platform-independent installation, versioning of software environments, and easy redeployment and reproducibility, Docker is an ideal candidate for the deployment of identical software stacks on different compute environments such as XSEDE and Amazon AWS. Cyverse's Discovery Environment also uses Docker for integrating its powerful, community-recommended software tools into CyVerse's production environment for public use. This paper will help users bring their tools into CyVerse DE which will not only allows users to integrate their tools with relative ease compared to the earlier method of tool deployment in DE but also help users to share their apps with collaborators and also release them for public use

Bringing your tools to CyVerse Discovery Environment using Docker [version 1; referees: 2 approved]

Docker has become a very popular container-based virtualization platform for software distribution that has revolutionized the way in which scientific software and software dependencies (software stacks) can be packaged, distributed, and deployed. Docker makes the complex and time-consuming installation procedures needed for scientific software a one-time process. Because it enables platform-independent installation, versioning of software environments, and easy redeployment and reproducibility, Docker is an ideal candidate for the deployment of identical software stacks on different compute environments such as XSEDE and Amazon AWS. CyVerse’s Discovery Environment also uses Docker for integrating its powerful, community-recommended software tools into CyVerse’s production environment for public use. This paper will help users bring their tools into CyVerse Discovery Environment (DE) which will not only allows users to integrate their tools with relative ease compared to the earlier method of tool deployment in DE but will also help users to share their apps with collaborators and release them for public use