Summary
Next‐generation sequencing technologies allow an almost exhaustive survey of the transcriptome, even in species with no available genome sequence. To produce a Unigene set representing most ...of the expressed genes of pea, 20 cDNA libraries produced from various plant tissues harvested at various developmental stages from plants grown under contrasting nitrogen conditions were sequenced. Around one billion reads and 100 Gb of sequence were de novo assembled. Following several steps of redundancy reduction, 46 099 contigs with N50 length of 1667 nt were identified. These constitute the ‘Caméor’ Unigene set. The high depth of sequencing allowed identification of rare transcripts and detected expression for approximately 80% of contigs in each library. The Unigene set is now available online (http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi), allowing (i) searches for pea orthologs of candidate genes based on gene sequences from other species, or based on annotation, (ii) determination of transcript expression patterns using various metrics, (iii) identification of uncharacterized genes with interesting patterns of expression, and (iv) comparison of gene ontology pathways between tissues. This resource has allowed identification of the pea orthologs of major nodulation genes characterized in recent years in model species, as a major step towards deciphering unresolved pea nodulation phenotypes. In addition to a remarkable conservation of the early transcriptome nodulation apparatus between pea and Medicago truncatula, some specific features were highlighted. The resource provides a reference for the pea exome, and will facilitate transcriptome and proteome approaches as well as SNP discovery in pea.
Significance Statement
From Mendel's discovery of the laws of genetics up to the advent of molecular biology, pea has been a valuable model for genetics and physiology. We present a comprehensive inventory of the expressed genes of pea in a readily searchable format. This resource strengthens pea as a model species and will facilitate searches for candidate gene sequences, microarray design, large‐scale proteomics studies, and identification of major genes in available mutant populations.
Coffee is a valuable beverage crop due to its characteristic flavor, aroma, and the stimulating effects of caffeine. We generated a high-quality draft genome of the species Coffea canephora, which ...displays a conserved chromosomal gene order among asterid angiosperms. Although it shows no sign of the whole-genome triplication identified in Solanaceae species such as tomato, the genome includes several species-specific gene family expansions, among them N-methyltransferases (NMTs) involved in caffeine production, defense-related genes, and alkaloid and flavonoid enzymes involved in secondary compound synthesis. Comparative analyses of caffeine NMTs demonstrate that these genes expanded through sequential tandem duplications independently of genes from cacao and tea, suggesting that caffeine in eudicots is of polyphyletic origin.
The combination of long reads and long-range information to produce genome assemblies is now accepted as a common standard. This strategy not only allows access to the gene catalogue of a given ...species but also reveals the architecture and organization of chromosomes, including complex regions such as telomeres and centromeres. The Brassica genus is not exempt, and many assemblies based on long reads are now available. The reference genome for Brassica napus, Darmor-bzh, which was published in 2014, was produced using short reads and its contiguity was extremely low compared with current assemblies of the Brassica genus.
Herein, we report the new long-read assembly of Darmor-bzh genome (Brassica napus) generated by combining long-read sequencing data and optical and genetic maps. Using the PromethION device and 6 flowcells, we generated ∼16 million long reads representing 93× coverage and, more importantly, 6× with reads longer than 100 kb. This ultralong-read dataset allows us to generate one of the most contiguous and complete assemblies of a Brassica genome to date (contig N50 > 10 Mb). In addition, we exploited all the advantages of the nanopore technology to detect modified bases and sequence transcriptomic data using direct RNA to annotate the genome and focus on resistance genes.
Using these cutting-edge technologies, and in particular by relying on all the advantages of the nanopore technology, we provide the most contiguous Brassica napus assembly, a resource that will be valuable to the Brassica community for crop improvement and will facilitate the rapid selection of agronomically important traits.
Abstract
Long-read sequencing currently provides sequences of several thousand base pairs. It is therefore possible to obtain complete transcripts, offering an unprecedented vision of the cellular ...transcriptome. However the literature lacks tools for de novo clustering of such data, in particular for Oxford Nanopore Technologies reads, because of the inherent high error rate compared to short reads. Our goal is to process reads from whole transcriptome sequencing data accurately and without a reference genome in order to reliably group reads coming from the same gene. This de novo approach is therefore particularly suitable for non-model species, but can also serve as a useful pre-processing step to improve read mapping. Our contribution both proposes a new algorithm adapted to clustering of reads by gene and a practical and free access tool that allows to scale the complete processing of eukaryotic transcriptomes. We sequenced a mouse RNA sample using the MinION device. This dataset is used to compare our solution to other algorithms used in the context of biological clustering. We demonstrate that it is the best approach for transcriptomics long reads. When a reference is available to enable mapping, we show that it stands as an alternative method that predicts complementary clusters.
Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the ...wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.
Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity ...of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies and tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing internal runs of T's. This bias is marked for runs of at least 15 T's, but is already detectable for runs of at least 9 T's and therefore concerns more than 20% of expressed transcripts in mouse brain and liver. Finally, we outline that bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of repeat-associated genes such as processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.
Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these ...agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means.
Root-knot nematodes (genus Meloidogyne) exhibit a diversity of reproductive modes ranging from obligatory sexual to fully asexual reproduction. Intriguingly, the most widespread and devastating ...species to global agriculture are those that reproduce asexually, without meiosis. To disentangle this surprising parasitic success despite the absence of sex and genetic exchanges, we have sequenced and assembled the genomes of three obligatory ameiotic and asexual Meloidogyne. We have compared them to those of relatives able to perform meiosis and sexual reproduction. We show that the genomes of ameiotic asexual Meloidogyne are large, polyploid and made of duplicated regions with a high within-species average nucleotide divergence of ~8%. Phylogenomic analysis of the genes present in these duplicated regions suggests that they originated from multiple hybridization events and are thus homoeologs. We found that up to 22% of homoeologous gene pairs were under positive selection and these genes covered a wide spectrum of predicted functional categories. To biologically assess functional divergence, we compared expression patterns of homoeologous gene pairs across developmental life stages using an RNAseq approach in the most economically important asexually-reproducing nematode. We showed that >60% of homoeologous gene pairs display diverged expression patterns. These results suggest a substantial functional impact of the genome structure. Contrasting with high within-species nuclear genome divergence, mitochondrial genome divergence between the three ameiotic asexuals was very low, signifying that these putative hybrids share a recent common maternal ancestor. Transposable elements (TE) cover a ~1.7 times higher proportion of the genomes of the ameiotic asexual Meloidogyne compared to the sexual relative and might also participate in their plasticity. The intriguing parasitic success of asexually-reproducing Meloidogyne species could be partly explained by their TE-rich composite genomes, resulting from allopolyploidization events, and promoting plasticity and functional divergence between gene copies in the absence of sex and meiosis.
Bracoviruses (BVs) from the Polydnaviridae family are symbiotic viruses used as biological weapons by parasitoid wasps to manipulate lepidopteran host physiology and induce parasitism success. BV ...particles are produced by wasp ovaries and injected along with the eggs into the caterpillar host body, where viral gene expression is necessary for wasp development. Recent sequencing of the proviral genome of Cotesia congregata BV (CcBV) identified 222 predicted virulence genes present on 35 proviral segments integrated into the wasp genome. To date, the expressions of only a few selected candidate virulence genes have been studied in the caterpillar host, and we lacked a global vision of viral gene expression. In this study, a large-scale transcriptomic analysis by 454 sequencing of two immune tissues (fat body and hemocytes) of parasitized Manduca sexta caterpillar hosts allowed the detection of expression of 88 CcBV genes expressed 24 h after the onset of parasitism. We linked the expression profiles of these genes to several factors, showing that different regulatory mechanisms control viral gene expression in the host. These factors include the presence of signal peptides in encoded proteins, diversification of promoter regions, and, more surprisingly, gene position on the proviral genome. Indeed, most genes for which expression could be detected are localized in particular proviral regions globally producing higher numbers of circles. Moreover, this polydnavirus (PDV) transcriptomic analysis also reveals that a majority of CcBV genes possess at least one intron and an arthropod transcription start site, consistent with an insect origin of these virulence genes.
Bracoviruses (BVs) are symbiotic polydnaviruses used by parasitoid wasps to manipulate lepidopteran host physiology, ensuring wasp offspring survival. To date, the expressions of only a few selected candidate BV virulence genes have been studied in caterpillar hosts. We performed a large-scale analysis of BV gene expression in two immune tissues of Manduca sexta caterpillars parasitized by Cotesia congregata wasps. Genes for which expression could be detected corresponded to genes localized in particular regions of the viral genome globally producing higher numbers of circles. Our study thus brings an original global vision of viral gene expression and paves the way to the determination of the regulatory mechanisms enabling the expression of BV genes in targeted organisms, such as major insect pests. In addition, we identify sequence features suggesting that most BV virulence genes were acquired from insect genomes.
Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made ...these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.
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