Summary
The TomExpress platform was developed to provide the tomato research community with a browser and integrated web tools for public RNA‐Seq data visualization and data mining. To avoid major ...biases that can result from the use of different mapping and statistical processing methods, RNA‐Seq raw sequence data available in public databases were mapped de novo on a unique tomato reference genome sequence and post‐processed using the same pipeline with accurate parameters. Following the calculation of the number of counts per gene in each RNA‐Seq sample, a communal global normalization method was applied to all expression values. This unifies the whole set of expression data and makes them comparable. A database was designed where each expression value is associated with corresponding experimental annotations. Sample details were manually curated to be easily understandable by biologists. To make the data easily searchable, a user‐friendly web interface was developed that provides versatile data mining web tools via on‐the‐fly generation of output graphics, such as expression bar plots, comprehensive in planta representations and heatmaps of hierarchically clustered expression data. In addition, it allows for the identification of co‐expressed genes and the visualization of correlation networks of co‐regulated gene groups. TomExpress provides one of the most complete free resources of publicly available tomato RNA‐Seq data, and allows for the immediate interrogation of transcriptional programs that regulate vegetative and reproductive development in tomato under diverse conditions. The design of the pipeline developed in this project enables easy updating of the database with newly published RNA‐Seq data, thereby allowing for continuous enrichment of the resource.
Significance Statement
After applying a bioinformatics and statistics pipeline, a unified TomExpress platform was developed to provide the tomato research community with a browser and integrated web tools for public RNA‐Seq data visualization and data mining via on‐the‐fly generation of output graphics such as expression bar plots, comprehensive in planta representations and heatmaps of hierarchically clustered expression data. TomExpress provides one of the most complete free resources of publicly available tomato RNA‐Seq data.
All-flesh tomato cultivars are devoid of locular gel and exhibit enhanced firmness and improved postharvest storage. Here, we show that SlMBP3 is a master regulator of locular tissue in tomato fruit ...and that a deletion at the gene locus underpins the All-flesh trait. Intriguingly, All-flesh varieties lack the deleterious phenotypes reported previously for SlMBP3 under-expressing lines and which preclude any potential commercial use. We resolve the causal factor for this phenotypic divergence through the discovery of a natural mutation at the SlAGL11 locus, a close homolog of SlMBP3. Misexpressing SlMBP3 impairs locular gel formation through massive transcriptomic reprogramming at initial phases of fruit development. SlMBP3 influences locule gel formation by controlling cell cycle and cell expansion genes, indicating that important components of fruit softening are determined at early pre-ripening stages. Our findings define potential breeding targets for improved texture in tomato and possibly other fleshy fruits.
The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, ...suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10(-4)) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.
Summary
As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. ...Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene‐specific ploidy‐regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA‐Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry‐sorted nuclei based on their DNA content. Here we show that cell‐based approaches to the study of RNA‐Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy‐specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.
Significance statement
RNA sequencing analysis of sorted endoreduplicated nuclei from tomato fruit pericarp tissue shows a global shift in gene expression due to ploidy, which ultimately influences data normalization and interpretation. In addition more than a thousand genes were found to be differentially expressed during the endoreduplication process, suggesting a ploidy‐ and/or cell size‐specific gene expression program.
transcriptome assembly of short reads is now a common step in expression analysis of organisms lacking a reference genome sequence. Several software packages are available to perform this task. Even ...if their results are of good quality it is still possible to improve them in several ways including redundancy reduction or error correction. Trinity and Oases are two commonly used
transcriptome assemblers. The contig sets they produce are of good quality. Still, their compaction (number of contigs needed to represent the transcriptome) and their quality (chimera and nucleotide error rates) can be improved.
We built a
RNA-Seq Assembly Pipeline (DRAP) which wraps these two assemblers (Trinity and Oases) in order to improve their results regarding the above-mentioned criteria. DRAP reduces from 1.3 to 15 fold the number of resulting contigs of the assemblies depending on the read set and the assembler used. This article presents seven assembly comparisons showing in some cases drastic improvements when using DRAP. DRAP does not significantly impair assembly quality metrics such are read realignment rate or protein reconstruction counts.
Transcriptome assembly is a challenging computational task even if good solutions are already available to end-users, these solutions can still be improved while conserving the overall representation and quality of the assembly. The
RNA-Seq Assembly Pipeline (DRAP) is an easy to use software package to produce compact and corrected transcript set. DRAP is free, open-source and available under GPL V3 license at http://www.sigenae.org/drap.
High-throughput sequencing technologies have offered in recent years new opportunities to study genome variations. These studies have mostly focused on single nucleotide polymorphisms, small ...insertions or deletions and on copy number variants. Other structural variants, such as large insertions or deletions, tandem duplications, translocations, and inversions are less well-studied, despite that some have an important impact on phenotypes. In the present study, we performed a large-scale survey of structural variants in cattle. We report the identification of 6,426 putative structural variants in cattle extracted from whole-genome sequence data of 62 bulls representing the three major French dairy breeds. These genomic variants affect DNA segments greater than 50 base pairs and correspond to deletions, inversions and tandem duplications. Out of these, we identified a total of 547 deletions and 410 tandem duplications which could potentially code for CNVs. Experimental validation was carried out on 331 structural variants using a novel high-throughput genotyping method. Out of these, 255 structural variants (77%) generated good quality genotypes and 191 (75%) of them were validated. Gene content analyses in structural variant regions revealed 941 large deletions removing completely one or several genes, including 10 single-copy genes. In addition, some of the structural variants are located within quantitative trait loci for dairy traits. This study is a pan-genome assessment of genomic variations in cattle and may provide a new glimpse into the bovine genome architecture. Our results may also help to study the effects of structural variants on gene expression and consequently their effect on certain phenotypes of interest.
Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects ...for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.
RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of ...transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome.
Transcriptome analysis based on a de novo assembly of next generation RNA sequences is now performed routinely in many laboratories. The generated results, including contig sequences, quantification ...figures, functional annotations and variation discovery outputs are usually bulky and quite diverse. This article presents a user oriented storage and visualisation environment permitting to explore the data in a top-down manner, going from general graphical views to all possible details. The software package is based on biomart, easy to install and populate with local data. The software package is available under the GNU General Public License (GPL) at http://bioinfo.genotoul.fr/RNAbrowse.
Cucumis melo displays a large diversity of horticultural groups with cantaloupe melon the most cultivated type. Using a combination of single-molecule sequencing, 10X Genomics link-reads, ...high-density optical and genetic maps, and chromosome conformation capture (Hi-C), we assembled a chromosome scale C. melo var. cantalupensis Charentais mono genome. Integration of RNA-seq, MeDip-seq, ChIP-seq, and Hi-C data revealed a widespread compartmentalization of the melon genome, segregating constitutive heterochromatin and euchromatin. Genome-wide comparative and evolutionary analysis between melon botanical groups identified Charentais mono genome increasingly more divergent from Harukei-3 (reticulatus), Payzawat (inodorus), and HS (ssp. agrestis) genomes. To assess the paleohistory of the Cucurbitaceae, we reconstructed the ancestral Cucurbitaceae karyotype and compared it to sequenced cucurbit genomes. In contrast to other species that experienced massive chromosome shuffling, melon has retained the ancestral genome structure. We provide comprehensive genomic resources and new insights in the diversity of melon horticultural groups and evolution of cucurbits.
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•We provide a chromosome scale C. melo var. cantalupensis Charentais mono genome•Epigenomic analysis revealed a widespread compartmentalization of the melon genome•We reconstructed the ancestral Cucurbitaceae karyotype•Melon has retained the ancestral Cucurbitaceae genome structure
Genomics; Omics; Plant biology; Plant evolution
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