Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing

<p>Abstract</p> <p>Background</p> <p>In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage.</p> <p>Results</p> <p>To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20–454 pyrosequencing runs of a cDNA library obtained from 2 week-old <it>Palomero Toluqueño </it>maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20–454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20–454 sequences and corresponding levels of gene expression.</p> <p>Conclusion</p> <p>A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.</p

ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

<p>Abstract</p> <p>Background</p> <p>In some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.</p> <p>Results</p> <p>We have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.</p> <p>Conclusion</p> <p>The ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at <url>http://wheat.pw.usda.gov/demos/ConservedPrimers/</url>.</p

Comprehensive prediction of novel microRNA targets in Arabidopsis thaliana

MicroRNAs (miRNAs) are 20–24 nt long endogenous non-coding RNAs that act as post-transcriptional regulators in metazoa and plants. Plant miRNA targets typically contain a single sequence motif with near-perfect complementarity to the miRNA. Here, we extended and applied the program RNAhybrid to identify novel miRNA targets in the complete annotated Arabidopsis thaliana transcriptome. RNAhybrid predicts the energetically most favorable miRNA:mRNA hybrids that are consistent with user-defined structural constraints. These were: (i) perfect base pairing of the duplex from nucleotide 8 to 12 counting from the 5′-end of the miRNA; (ii) loops with a maximum length of one nucleotide in either strand; (iii) bulges with no more than one nucleotide in size; and (iv) unpaired end overhangs not longer than two nucleotides. G:U base pairs are not treated as mismatches, but contribute less favorable to the overall free energy. The resulting hybrids were filtered according to their minimum free energy, resulting in an overall prediction of more than 600 novel miRNA targets. The specificity and signal-to-noise ratio of the prediction was assessed with either randomized miRNAs or randomized target sequences as negative controls. Our results are in line with recent observations that the majority of miRNA targets are not transcription factors

Global Analysis of Proline-Rich Tandem Repeat Proteins Reveals Broad Phylogenetic Diversity in Plant Secretomes

Cell walls, constructed by precisely choreographed changes in the plant secretome, play critical roles in plant cell physiology and development. Along with structural polysaccharides, secreted proline-rich Tandem Repeat Proteins (TRPs) are important for cell wall function, yet the evolutionary diversity of these structural TRPs remains virtually unexplored. Using a systems-level computational approach to analyze taxonomically diverse plant sequence data, we identified 31 distinct Pro-rich TRP classes targeted for secretion. This analysis expands upon the known phylogenetic diversity of extensins, the most widely studied class of wall structural proteins, and demonstrates that extensins evolved before plant vascularization. Our results also show that most Pro-rich TRP classes have unexpectedly restricted evolutionary distributions, revealing considerable differences in plant secretome signatures that define unexplored diversity

TRAPID : an efficient online tool for the functional and comparative analysis of de novo RNA-Seq transcriptomes

Transcriptome analysis through next-generation sequencing technologies allows the generation of detailed gene catalogs for non-model species, at the cost of new challenges with regards to computational requirements and bioinformatics expertise. Here, we present TRAPID, an online tool for the fast and efficient processing of assembled RNA-Seq transcriptome data, developed to mitigate these challenges. TRAPID offers high-throughput open reading frame detection, frameshift correction and includes a functional, comparative and phylogenetic toolbox, making use of 175 reference proteomes. Benchmarking and comparison against state-of-the-art transcript analysis tools reveals the efficiency and unique features of the TRAPID system

The TIGR Rice Genome Annotation Resource: improvements and new features

In The Institute for Genomic Research Rice Genome Annotation project (), we have continued to update the rice genome sequence with new data and improve the quality of the annotation. In our current release of annotation (Release 4.0; January 12, 2006), we have identified 42 653 non-transposable element-related genes encoding 49 472 gene models as a result of the detection of alternative splicing. We have refined our identification methods for transposable element-related genes resulting in 13 237 genes that are related to transposable elements. Through incorporation of multiple transcript and proteomic expression data sets, we have been able to annotate 24 799 genes (31 739 gene models), representing ∼50% of the total gene models, as expressed in the rice genome. All structural and functional annotation is viewable through our Rice Genome Browser which currently supports 59 tracks. Enhanced data access is available through web interfaces, FTP downloads and a Data Extractor tool developed in order to support discrete dataset downloads

The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair

Background: The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants.Description: These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the " Barley Genome version 0.05" database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair.Conclusions: The presented database provides information about the Arabidopsis genes involved in DNA replication and repair, their expression patterns and models of protein interactions. It was designed and established to provide an open-access tool for the identification of monocot homologs of known Arabidopsis genes that are responsible for DNA-related processes. The barley genes identified in the project are currently being analysed to validate their function

TriMEDB: A database to integrate transcribed markers and facilitate genetic studies of the tribe Triticeae

<p>Abstract</p> <p>Background</p> <p>The recent rapid accumulation of sequence resources of various crop species ensures an improvement in the genetics approach, including quantitative trait loci (QTL) analysis as well as the holistic population analysis and association mapping of natural variations. Because the tribe Triticeae includes important cereals such as wheat and barley, integration of information on the genetic markers in these crops should effectively accelerate map-based genetic studies on Triticeae species and lead to the discovery of key loci involved in plant productivity, which can contribute to sustainable food production. Therefore, informatics applications and a semantic knowledgebase of genome-wide markers are required for the integration of information on and further development of genetic markers in wheat and barley in order to advance conventional marker-assisted genetic analyses and population genomics of Triticeae species.</p> <p>Description</p> <p>The Triticeae mapped expressed sequence tag (EST) database (TriMEDB) provides information, along with various annotations, regarding mapped cDNA markers that are related to barley and their homologues in wheat. The current version of TriMEDB provides map-location data for barley and wheat ESTs that were retrieved from 3 published barley linkage maps (the barley single nucleotide polymorphism database of the Scottish Crop Research Institute, the barley transcript map of Leibniz Institute of Plant Genetics and Crop Plant Research, and HarvEST barley ver. 1.63) and 1 diploid wheat map. These data were imported to CMap to allow the visualization of the map positions of the ESTs and interrelationships of these ESTs with public gene models and representative cDNA sequences. The retrieved cDNA sequences corresponding to each EST marker were assigned to the rice genome to predict an exon-intron structure. Furthermore, to generate a unique set of EST markers in Triticeae plants among the public domain, 3472 markers were assembled to form 2737 unique marker groups as contigs. These contigs were applied for pairwise comparison among linkage maps obtained from different EST map resources.</p> <p>Conclusion</p> <p>TriMEDB provides information regarding transcribed genetic markers and functions as a semantic knowledgebase offering an informatics facility for the acceleration of QTL analysis and for population genetics studies of Triticeae.</p

Evolution of xyloglucan-related genes in green plants

<p>Abstract</p> <p>Background</p> <p>The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.</p> <p>Results</p> <p>In this work, we used a comparative genomic approach to obtain new insights into the evolution of the xyloglucan-related enzymatic machinery in green plants. Detailed phylogenetic analyses were done for enzymes involved in xyloglucan synthesis (xyloglucan transglycosylase/hydrolase, α-xylosidase, β-galactosidase, β-glucosidase and α-fucosidase) and mobilization/degradation (β-(1→4)-glucan synthase, α-fucosyltransferases, β-galactosyltransferases and α-xylosyl transferase) based on 12 fully sequenced genomes and expressed sequence tags from 29 species of green plants. Evidence from Chlorophyta and Streptophyta green algae indicated that part of the Embryophyta xyloglucan-related machinery evolved in an aquatic environment, before land colonization. Streptophyte algae have at least three enzymes of the xyloglucan machinery: xyloglucan transglycosylase/hydrolase, β-(1→4)-glucan synthase from the celullose synthase-like C family and α-xylosidase that is also present in chlorophytes. Interestingly, gymnosperm sequences orthologs to xyloglucan transglycosylase/hydrolases with exclusively hydrolytic activity were also detected, suggesting that such activity must have emerged within the last common ancestor of spermatophytes. There was a positive correlation between the numbers of founder genes within each gene family and the complexity of the plant cell wall.</p> <p>Conclusions</p> <p>Our data support the idea that a primordial xyloglucan-like polymer emerged in streptophyte algae as a pre-adaptation that allowed plants to subsequently colonize terrestrial habitats. Our results also provide additional evidence that charophycean algae and land plants are sister groups.</p