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April 21, 2020  |  

Genetic basis for the establishment of endosymbiosis in Paramecium.

The single-celled ciliate Paramecium bursaria is an indispensable model for investigating endosymbiosis between protists and green-algal symbionts. To elucidate the mechanism of this type of endosymbiosis, we combined PacBio and Illumina sequencing to assemble a high-quality and near-complete macronuclear genome of P. bursaria. The genomic characteristics and phylogenetic analyses indicate that P. bursaria is the basal clade of the Paramecium genus. Through comparative genomic analyses with its close relatives, we found that P. bursaria encodes more genes related to nitrogen metabolism and mineral absorption, but encodes fewer genes involved in oxygen binding and N-glycan biosynthesis. A comparison of the transcriptomic profiles between P. bursaria with and without endosymbiotic Chlorella showed differential expression of a wide range of metabolic genes. We selected 32 most differentially expressed genes to perform RNA interference experiment in P. bursaria, and found that P. bursaria can regulate the abundance of their symbionts through glutamine supply. This study provides novel insights into Paramecium evolution and will extend our knowledge of the molecular mechanism for the induction of endosymbiosis between P. bursaria and green algae.

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April 21, 2020  |  

Diploid Genome Assembly of the Wine Grape Carménère.

In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.Copyright © 2019 Minio et al.

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April 21, 2020  |  

The role of genomic structural variation in the genetic improvement of polyploid crops

Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence–absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence–absence variation and copy-number variation, we propose that homeologous exchanges constitute a third major form of genomic structural variation in polyploids. Homeologous exchanges involve the replacement of one genomic segment by a similar copy from another genome or ancestrally duplicated region, and are known to be extremely common in polyploids. Detecting all kinds of genomic structural variation is challenging, but recent advances such as optical mapping and long-read sequencing offer potential strategies to help identify structural variants even in complex polyploid genomes. All three major types of genomic structural variation (presence–absence, copy-number, and homeologous exchange) are now known to influence phenotypes in crop plants, with examples of flowering time, frost tolerance, and adaptive and agronomic traits. In this review, we summarize the challenges of genome analysis in polyploid crops, describe the various types of genomic structural variation and the genomics technologies and data that can be used to detect them, and collate information produced to date related to the impact of genomic structural variation on crop phenotypes. We highlight the importance of genomic structural variation for the future genetic improvement of polyploid crops.

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April 21, 2020  |  

Genome of the Komodo dragon reveals adaptations in the cardiovascular and chemosensory systems of monitor lizards.

Monitor lizards are unique among ectothermic reptiles in that they have high aerobic capacity and distinctive cardiovascular physiology resembling that of endothermic mammals. Here, we sequence the genome of the Komodo dragon Varanus komodoensis, the largest extant monitor lizard, and generate a high-resolution de novo chromosome-assigned genome assembly for V. komodoensis using a hybrid approach of long-range sequencing and single-molecule optical mapping. Comparing the genome of V. komodoensis with those of related species, we find evidence of positive selection in pathways related to energy metabolism, cardiovascular homoeostasis, and haemostasis. We also show species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and other lizard lineages. Together, these evolutionary signatures of adaptation reveal the genetic underpinnings of the unique Komodo dragon sensory and cardiovascular systems, and suggest that selective pressure altered haemostasis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. The Komodo dragon genome is an important resource for understanding the biology of monitor lizards and reptiles worldwide.

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April 21, 2020  |  

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020  |  

Single-molecule real-time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.).

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene ß-cyclase, ß-carotene hydroxylase, zeaxanthin epoxidase and capsanthin-capsorubin synthase (CCS) genes, in 94 pepper accessions by single-molecule real-time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra-performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.

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April 21, 2020  |  

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020  |  

The complete genome sequence of the denitrifying bacterium Marinobacter sp. Arc7-DN-1 isolated from Arctic Ocean sediment

The general features and genome characteristics of the denitrifying bacterium Marinobacter sp. Arc7-DN-1, isolated from Arctic Ocean sediment, are described. Marinobacter sp. Arc7-DN-1 uses NO3- or NH4+ as the sole nitrogen source to grow at low temperatures. The strain can grow at a wide range of temperatures (0–30?°C) and NaCl concentration (15–90‰). The genome has one circular chromosome of 4,300,456?bp (57.64?mol%?G?+?C content), consisting of 4012 coding genes, including 50 tRNAs and three rRNA operons as 16S-23S-5S rRNA. On the basis of the KEGG analysis, strain Arc7-DN-1 encodes 43 proteins related to nitrogen metabolism, including a complete denitrifying pathway and an assimilatory nitrate reduction pathway.

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April 21, 2020  |  

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

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April 21, 2020  |  

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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April 21, 2020  |  

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.

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April 21, 2020  |  

A reference-grade wild soybean genome.

Efficient crop improvement depends on the application of accurate genetic information contained in diverse germplasm resources. Here we report a reference-grade genome of wild soybean accession W05, with a final assembled genome size of 1013.2?Mb and a contig N50 of 3.3?Mb. The analytical power of the W05 genome is demonstrated by several examples. First, we identify an inversion at the locus determining seed coat color during domestication. Second, a translocation event between chromosomes 11 and 13 of some genotypes is shown to interfere with the assignment of QTLs. Third, we find a region containing copy number variations of the Kunitz trypsin inhibitor (KTI) genes. Such findings illustrate the power of this assembly in the analysis of large structural variations in soybean germplasm collections. The wild soybean genome assembly has wide applications in comparative genomic and evolutionary studies, as well as in crop breeding and improvement programs.

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April 21, 2020  |  

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.

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April 21, 2020  |  

Comparative Genomics of Thiohalobacter thiocyanaticus HRh1T and Guyparkeria sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Gammaproteobacteria Capable of Using Thiocyanate as Energy Source.

The genomes of Thiohalobacter thiocyanaticus and Guyparkeria (formerly known as Halothiobacillus) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the “cyanate pathway”, have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the “cyanate pathway” in members of the haloalkaliphilic SOB of the genus Thioalkalivibrio. However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the Thioalkalivibrio enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system Cus is present instead of Cop and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing Thioalkalivibrio species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of Thiohalobacter includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of Guyparkeria sp. SCN-R1 was more similar to that of members of the Thiomicrospira-Hydrogenovibrio group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of Thiohalobacter is the presence of a Na+-dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the Gammaproteobacteria developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.

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