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De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants.

ABSTRACT: BACKGROUND:The shift from cross-fertilization to predominant self-fertilization is among the most common evolutionary transitions in the reproductive biology of flowering plants. Increased inbreeding has important consequences for floral morphology, population genetic structure and genome evolution. The transition to selfing is usually characterized by a marked reduction in flower size and the loss of traits involved in pollinator attraction and the avoidance of self-fertilization. Here, we use short-read sequencing to assemble, de novo, the floral transcriptomes of three genotypes of Eichhornia paniculata, including an outcrosser and two genotypes from independently derived selfers, and a single genotype of the sister species E. paradoxa. By sequencing mRNA from tissues sampled at various stages of flower development, our goal was to sequence and assemble the floral transcriptome and identify differential patterns of gene expression. RESULTS:Our 24 Mbp assembly resulted in ~27,000 contigs that averaged ~900 bp in length. All four genotypes had highly correlated gene expression, but the three E. paniculata genotypes were more correlated with one another than each was to E. paradoxa. Our analysis identified 269 genes associated with floral development, 22 of which were differentially expressed in selfing lineages relative to the outcrosser. Many of the differentially expressed genes affect floral traits commonly altered in selfing plants and these represent a set of potential candidate genes for investigating the evolution of the selfing syndrome. CONCLUSIONS:Our study is among the first to demonstrate the use of Illumina short read sequencing for de novo transcriptome assembly in non-model species, and the first to implement this technology for comparing floral transcriptomes in outcrossing and selfing plants.

SUBMITTER: Ness RW

PROVIDER: S-EPMC3128866 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants.

Ness Rob W RW Siol Mathieu M Barrett Spencer C H SC

BMC genomics 20110607

<h4>Background</h4>The shift from cross-fertilization to predominant self-fertilization is among the most common evolutionary transitions in the reproductive biology of flowering plants. Increased inbreeding has important consequences for floral morphology, population genetic structure and genome evolution. The transition to selfing is usually characterized by a marked reduction in flower size and the loss of traits involved in pollinator attraction and the avoidance of self-fertilization. Here, ...[more]

PMID: 21649902

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Project description:BackgroundThe ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target's exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly.MethodsTo assess whether de novo transcriptome assemblies can be used to determine a protein's sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits. We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits' sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement.ResultsFor the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey.ConclusionsIn summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.

Project description:BACKGROUND:Nicotiana benthamiana has been widely used in laboratories around the world for studying plant-pathogen interactions and posttranscriptional gene expression silencing. Yet the exploration of its transcriptome has lagged behind due to the lack of both adequate sequence information and genome-wide analysis tools, such as DNA microarrays. Despite the increasing use of high-throughput sequencing technologies, the DNA microarrays still remain a popular gene expression tool, because they are cheaper and less demanding regarding bioinformatics skills and computational effort. RESULTS:We designed a gene expression microarray with 103,747 60-mer probes, based on two recently published versions of N. benthamiana transcriptome (v.3 and v.5). Both versions were reconstructed from RNA-Seq data of non-strand-specific pooled-tissue libraries, so we defined the sense strand of the contigs prior to designing the probe. To accomplish this, we combined a homology search against Arabidopsis thaliana proteins and hybridization to a test 244k microarray containing pairs of probes, which represented individual contigs. We identified the sense strand in 106,684 transcriptome contigs and used this information to design an Nb-105k microarray on an Agilent eArray platform. Following hybridization of RNA samples from N. benthamiana roots and leaves we demonstrated that the new microarray had high specificity and sensitivity for detection of differentially expressed transcripts. We also showed that the data generated with the Nb-105k microarray may be used to identify incorrectly assembled contigs in the v.5 transcriptome, by detecting inconsistency in the gene expression profiles, which is indicated using multiple microarray probes that match the same v.5 primary transcripts. CONCLUSIONS:We provided a complete design of an oligonucleotide microarray that may be applied to the research of N. benthamiana transcriptome. This, in turn, will allow the N. benthamiana research community to take full advantage of microarray capabilities for studying gene expression in this plant. Additionally, by defining the sense orientation of over 106,000 contigs, we substantially improved the functional information on the N. benthamiana transcriptome. The simple hybridization-based approach for detecting the sense orientation of computationally assembled sequences can be used for updating the transcriptomes of other non-model organisms, including cases where no significant homology to known proteins exists.

Project description:BackgroundNicotiana benthamiana has been widely used for transient gene expression assays and as a model plant in the study of plant-microbe interactions, lipid engineering and RNA silencing pathways. Assembling the sequence of its transcriptome provides information that, in conjunction with the genome sequence, will facilitate gaining insight into the plant's capacity for high-level transient transgene expression, generation of mobile gene silencing signals, and hyper-susceptibility to viral infection.Methodology/resultsRNA-seq libraries from 9 different tissues were deep sequenced and assembled, de novo, into a representation of the transcriptome. The assembly, of 16GB of sequence, yielded 237,340 contigs, clustering into 119,014 transcripts (unigenes). Between 80 and 85% of reads from all tissues could be mapped back to the full transcriptome. Approximately 63% of the unigenes exhibited a match to the Solgenomics tomato predicted proteins database. Approximately 94% of the Solgenomics N. benthamiana unigene set (16,024 sequences) matched our unigene set (119,014 sequences). Using homology searches we identified 31 homologues that are involved in RNAi-associated pathways in Arabidopsis thaliana, and show that they possess the domains characteristic of these proteins. Of these genes, the RNA dependent RNA polymerase gene, Rdr1, is transcribed but has a 72 nt insertion in exon1 that would cause premature termination of translation. Dicer-like 3 (DCL3) appears to lack both the DEAD helicase motif and second dsRNA binding motif, and DCL2 and AGO4b have unexpectedly high levels of transcription.ConclusionsThe assembled and annotated representation of the transcriptome and list of RNAi-associated sequences are accessible at www.benthgenome.com alongside a draft genome assembly. These genomic resources will be very useful for further study of the developmental, metabolic and defense pathways of N. benthamiana and in understanding the mechanisms behind the features which have made it such a well-used model plant.

Dataset Information

De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants.

Publications

De novo sequence assembly and characterization of the floral transcriptome in cross- and self-fertilizing plants.

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