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July 7, 2019

NOVOPlasty: de novo assembly of organelle genomes from whole genome data.

The evolution in next-generation sequencing (NGS) technology has led to the development of many different assembly algorithms, but few of them focus on assembling the organelle genomes. These genomes are used in phylogenetic studies, food identification and are the most deposited eukaryotic genomes in GenBank. Producing organelle genome assembly from whole genome sequencing (WGS) data would be the most accurate and least laborious approach, but a tool specifically designed for this task is lacking. We developed a seed-and-extend algorithm that assembles organelle genomes from whole genome sequencing (WGS) data, starting from a related or distant single seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina data sets where it outperforms known assemblers in assembly accuracy and coverage. In our benchmark, NOVOPlasty assembled all tested circular genomes in less than 30 min with a maximum memory requirement of 16 GB and an accuracy over 99.99%. In conclusion, NOVOPlasty is the sole de novo assembler that provides a fast and straightforward extraction of the extranuclear genomes from WGS data in one circular high quality contig. The software is open source and can be downloaded at https://github.com/ndierckx/NOVOPlasty.

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July 7, 2019

Draft genome assembly and annotation of Glycyrrhiza uralensis, a medicinal legume.

Chinese liquorice/licorice (Glycyrrhiza uralensis) is a leguminous plant species whose roots and rhizomes have been widely used as a herbal medicine and natural sweetener. Whole-genome sequencing is essential for gene discovery studies and molecular breeding in liquorice. Here, we report a draft assembly of the approximately 379-Mb whole-genome sequence of strain 308-19 of G. uralensis; this assembly contains 34 445 predicted protein-coding genes. Comparative analyses suggested well-conserved genomic components and collinearity of gene loci (synteny) between the genome of liquorice and those of other legumes such as Medicago and chickpea. We observed that three genes involved in isoflavonoid biosynthesis, namely, 2-hydroxyisoflavanone synthase (CYP93C), 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase/isoflavone 4′-O-methyltransferase (HI4OMT) and isoflavone-7-O-methyltransferase (7-IOMT) formed a cluster on the scaffold of the liquorice genome and showed conserved microsynteny with Medicago and chickpea. Based on the liquorice genome annotation, we predicted genes in the P450 and UDP-dependent glycosyltransferase (UGT) superfamilies, some of which are involved in triterpenoid saponin biosynthesis, and characterised their gene expression with the reference genome sequence. The genome sequencing and its annotations provide an essential resource for liquorice improvement through molecular breeding and the discovery of useful genes for engineering bioactive components through synthetic biology approaches.© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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July 7, 2019

The comparative landscape of duplications in Heliconius melpomene and Heliconius cydno.

Gene duplications can facilitate adaptation and may lead to interpopulation divergence, causing reproductive isolation. We used whole-genome resequencing data from 34 butterflies to detect duplications in two Heliconius species, Heliconius cydno and Heliconius melpomene. Taking advantage of three distinctive signals of duplication in short-read sequencing data, we identified 744 duplicated loci in H. cydno and H. melpomene and evaluated the accuracy of our approach using single-molecule sequencing. We have found that duplications overlap genes significantly less than expected at random in H. melpomene, consistent with the action of background selection against duplicates in functional regions of the genome. Duplicate loci that are highly differentiated between H. melpomene and H. cydno map to four different chromosomes. Four duplications were identified with a strong signal of divergent selection, including an odorant binding protein and another in close proximity with a known wing colour pattern locus that differs between the two species. Heredity advance online publication, 7 December 2016; doi:10.1038/hdy.2016.107.

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July 7, 2019

Draft genome sequence of Mentha longifolia (L.) and development of resources for mint cultivar improvement.

The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. Desirable oil characteristics and resistance to the fungal disease Verticillium wilt are top priorities for the mint industry. However, cultivated mints have complex polyploid genomes and are sterile. Breeding efforts, therefore, require the development of genomic resources for fertile mint species. Here, we present draft de novo genome and plastome assemblies for a wilt-resistant South African accession of Mentha longifolia (L.) Huds., a diploid species ancestral to cultivated peppermint and spearmint. The 353 Mb genome contains 35 597 predicted protein-coding genes, including 292 disease resistance gene homologs, and nine genes determining essential oil characteristics. A genetic linkage map ordered 1397 genome scaffolds on 12 pseudochromosomes. More than two million simple sequence repeats were identified, which will facilitate molecular marker development. The M. longifolia genome is a valuable resource for both metabolic engineering and molecular breeding. This is exemplified by employing the genome sequence to clone and functionally characterize the promoters in a peppermint cultivar, and demonstrating the utility of a glandular trichome-specific promoter to increase expression of a biosynthetic gene, thereby modulating essential oil composition. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Complete genome sequence of Pseudomonas brassicacearum strain L13-6-12, a biological control agent from the rhizosphere of potato

Pseudomonas brassicacearum strain L13-6-12 is a rhizosphere colonizer of potato, lettuce and sugar beet. Previous studies have shown that this motile, Gram-negative, non-sporulating bacterium is an effective biocontrol agent against different phytopathogens. Here, we announce and describe the complete genome sequence of P. brassicacearum L13-6-12 consisting of a single 6.7 Mb circular chromosome that consists of 5773 protein coding genes and 85 RNA-only encoding genes. Genome analysis revealed genes encoding specialized functions for pathogen suppression, thriving in the rhizosphere and interacting with eukaryotic organisms.

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July 7, 2019

Competition assays and physiological experiments of soil and phyllosphere yeasts identify Candida subhashii as a novel antagonist of filamentous fungi.

While recent advances in next generation sequencing technologies have enabled researchers to readily identify countless microbial species in soil, rhizosphere, and phyllosphere microbiomes, the biological functions of the majority of these species are unknown. Functional studies are therefore urgently needed in order to characterize the plethora of microorganisms that are being identified and to point out species that may be used for biotechnology or plant protection. Here, we used a dual culture assay and growth analyses to characterise yeasts (40 different isolates) and their antagonistic effect on 16 filamentous fungi; comprising plant pathogens, antagonists, and saprophytes.Overall, this competition screen of 640 pairwise combinations revealed a broad range of outcomes, ranging from small stimulatory effects of some yeasts up to a growth inhibition of more than 80% by individual species. On average, yeasts isolated from soil suppressed filamentous fungi more strongly than phyllosphere yeasts and the antagonistic activity was a species-/isolate-specific property and not dependent on the filamentous fungus a yeast was interacting with. The isolates with the strongest antagonistic activity were Metschnikowia pulcherrima, Hanseniaspora sp., Cyberlindnera sargentensis, Aureobasidium pullulans, Candida subhashii, and Pichia kluyveri. Among these, the soil yeasts (C. sargentensis, A. pullulans, C. subhashii) assimilated and/or oxidized more di-, tri- and tetrasaccharides and organic acids than yeasts from the phyllosphere. Only the two yeasts C. subhashii and M. pulcherrima were able to grow with N-acetyl-glucosamine as carbon source.The competition assays and physiological experiments described here identified known antagonists that have been implicated in the biological control of plant pathogenic fungi in the past, but also little characterised species such as C. subhashii. Overall, soil yeasts were more antagonistic and metabolically versatile than yeasts from the phyllosphere. Noteworthy was the strong antagonistic activity of the soil yeast C. subhashii, which had so far only been described from a clinical sample and not been studied with respect to biocontrol. Based on binary competition assays and growth analyses (e.g., on different carbon sources, growth in root exudates), C. subhashii was identified as a competitive and antagonistic soil yeast with potential as a novel biocontrol agent against plant pathogenic fungi.

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July 7, 2019

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data.

The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read correction and de novo assembly using Sprai. Organelle-PBA completes the assembly process with the additional step of scaffolding by SSPACE-LongRead. The program then detects the chloroplast inverted repeats and reassembles and re-orients the assembly based on the organelle origin of the reference. We have evaluated the performance of the software using PacBio reads from different species, read coverage, and reference genomes. Finally, we present the assembly of two novel chloroplast genomes from the species Picea glauca (Pinaceae) and Sinningia speciosa (Gesneriaceae).Organelle-PBA is an easy-to-use Perl-based software pipeline that was written specifically to assemble mitochondrial and chloroplast genomes from whole genome PacBio reads. The program is available at https://github.com/aubombarely/Organelle_PBA .

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July 7, 2019

Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

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July 7, 2019

De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species.

Para rubber tree (Hevea brasiliensis) is an important economic species as it is the sole commercial producer of high-quality natural rubber. Here, we report a de novo hybrid assembly of BPM24 accession, which exhibits resistance to major fungal pathogens in Southeast Asia. Deep-coverage 454/Illumina short-read and Pacific Biosciences (PacBio) long-read sequence data were acquired to generate a preliminary draft, which was subsequently scaffolded using a long-range “Chicago” technique to obtain a final assembly of 1.26?Gb (N50?=?96.8?kb). The assembled genome contains 69.2% repetitive sequences and has a GC content of 34.31%. Using a high-density SNP-based genetic map, we were able to anchor 28.9% of the genome assembly (363?Mb) associated with over two thirds of the predicted protein-coding genes into rubber tree’s 18 linkage groups. These genetically anchored sequences allowed comparative analyses of the intragenomic homeologous synteny, providing the first concrete evidence to demonstrate the presence of paleotetraploidy in Hevea species. Additionally, the degree of macrosynteny conservation observed between rubber tree and cassava strongly supports the hypothesis that the paleotetraploidization event took place prior to the divergence of the Hevea and Manihot species.

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July 7, 2019

GenBank.

GenBank(®) (www.ncbi.nlm.nih.gov/genbank/) is a comprehensive database that contains publicly available nucleotide sequences for 370 000 formally described species. These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or the NCBI Submission Portal. GenBank staff assign accession numbers upon data receipt. Daily data exchange with the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through the NCBI Nucleotide database, which links to related information such as taxonomy, genomes, protein sequences and structures, and biomedical journal literature in PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. Recent updates include changes to policies regarding sequence identifiers, an improved 16S submission wizard, targeted loci studies, the ability to submit methylation and BioNano mapping files, and a database of anti-microbial resistance genes. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

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July 7, 2019

Identification of symmetrical RNA editing events in the mitochondria of Salvia miltiorrhiza by strand-specific RNA sequencing.

Salvia miltiorrhiza is one of the most widely-used medicinal plants. Here, we systematically analyzed the RNA editing events in its mitochondria. We developed a pipeline using REDItools to predict RNA editing events from stand-specific RNA-Seq data. The predictions were validated using reverse transcription, RT-PCR amplification and Sanger sequencing experiments. Putative sequences motifs were characterized. Comparative analyses were carried out between S. miltiorrhiza, Arabidopsis thaliana and Oryza sativa. We discovered 1123 editing sites, including 225 “C to U” sites in the protein-coding regions. Fourteen of sixteen (87.5%) sites were validated. Three putative DNA motifs were identified around the predicted sites. The nucleotides on both strands at 115 of the 225 sites had undergone RNA editing, which we called symmetrical RNA editing (SRE). Four of six these SRE sites (66.7%) were experimentally confirmed. Re-examination of strand-specific RNA-Seq data from A. thaliana and O. sativa identified 327 and 369 SRE sites respectively. 78, 20 and 13 SRE sites were found to be conserved among A. thaliana, O. sativa and S. miltiorrhiza respectively. This study provides a comprehensive picture of RNA editing events in the mitochondrial genome of S. miltiorrhiza. We identified SREs for the first time, which may represent a universal phenomenon.

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July 7, 2019

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephulotus folliculnris, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identi- fied genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. folliculnris and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.

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July 7, 2019

Complex modular architecture around a simple toolkit of wing pattern genes

Identifying the genomic changes that control morphological variation and understanding how they generate diversity is a major goal of evolutionary biology. In Heliconius butterflies, a small number of genes control the development of diverse wing colour patterns. Here, we used full-genome sequencing of individuals across the Heliconius erato radiation and closely related species to characterize genomic variation associated with wing pattern diversity. We show that variation around colour pattern genes is highly modular, with narrow genomic intervals associated with specific differences in colour and pattern. This modular architecture explains the diversity of colour patterns and provides a flexible mechanism for rapid morphological diversification.

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July 7, 2019

The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two ‘types’, G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for “dead-end” trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

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