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April 21, 2020  |  

Complete Whole-Genome Sequences of Two Raoultella terrigena Strains, NCTC 13097 and NCTC 13098, Isolated from Human Cases.

Raoultella terrigena is a bacterial species associated with soil and aquatic environments; however, sporadic cases of opportunistic disease in humans have been reported. Here, we report the first two complete genome sequences from clinical strains isolated from human sources that have been deposited in the National Collection of Type Cultures (NCTC). © Crown copyright 2019.


April 21, 2020  |  

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Plantibacter flavus, Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens Endophytes Provide Host-Specific Growth Promotion of Arabidopsis thaliana, Basil, Lettuce, and Bok Choy Plants.

A collection of bacterial endophytes isolated from stem tissues of plants growing in soils highly contaminated with petroleum hydrocarbons were screened for plant growth-promoting capabilities. Twenty-seven endophytic isolates significantly improved the growth of Arabidopsis thaliana plants in comparison to that of uninoculated control plants. The five most beneficial isolates, one strain each of Curtobacterium herbarum, Paenibacillus taichungensis, and Rhizobium selenitireducens and two strains of Plantibacter flavus were further examined for growth promotion in Arabidopsis, lettuce, basil, and bok choy plants. Host-specific plant growth promotion was observed when plants were inoculated with the five bacterial strains. P. flavus strain M251 increased the total biomass and total root length of Arabidopsis plants by 4.7 and 5.8 times, respectively, over that of control plants and improved lettuce and basil root growth, while P. flavus strain M259 promoted Arabidopsis shoot and root growth, lettuce and basil root growth, and bok choy shoot growth. A genome comparison between P. flavus strains M251 and M259 showed that both genomes contain up to 70 actinobacterial putative plant-associated genes and genes involved in known plant-beneficial pathways, such as those for auxin and cytokinin biosynthesis and 1-aminocyclopropane-1-carboxylate deaminase production. This study provides evidence of direct plant growth promotion by Plantibacter flavusIMPORTANCE The discovery of new plant growth-promoting bacteria is necessary for the continued development of biofertilizers, which are environmentally friendly and cost-efficient alternatives to conventional chemical fertilizers. Biofertilizer effects on plant growth can be inconsistent due to the complexity of plant-microbe interactions, as the same bacteria can be beneficial to the growth of some plant species and neutral or detrimental to others. We examined a set of bacterial endophytes isolated from plants growing in a unique petroleum-contaminated environment to discover plant growth-promoting bacteria. We show that strains of Plantibacter flavus exhibit strain-specific plant growth-promoting effects on four different plant species.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Hybrid Sequencing of Full-Length cDNA Transcripts of the Medicinal Plant Scutellaria baicalensis.

Scutellaria baicalensis is a well-known medicinal plant that produces biologically active flavonoids, such as baicalin, baicalein, and wogonin. Pharmacological studies have shown that these compounds have anti-inflammatory, anti-bacterial, and anti-cancer activities. Therefore, it is of great significance to investigate the genetic information of S. baicalensis, particularly the genes related to the biosynthetic pathways of these compounds. Here, we constructed the full-length transcriptome of S. baicalensis using a hybrid sequencing strategy and acquired 338,136 full-length sequences, accounting for 93.3% of the total reads. After the removal of redundancy and correction with Illumina short reads, 75,785 nonredundant transcripts were generated, among which approximately 98% were annotated with significant hits in the protein databases, and 11,135 sequences were classified as lncRNAs. Differentially expressed gene (DEG) analysis showed that most of the genes related to flavonoid biosynthesis were highly expressed in the roots, consistent with previous reports that the flavonoids were mainly synthesized and accumulated in the roots of S. baicalensis. By constructing unique transcription models, a total of 44,071 alternative splicing (AS) events were identified, with intron retention (IR) accounting for the highest proportion (44.5%). A total of 94 AS events were present in five key genes related to flavonoid biosynthesis, suggesting that AS may play important roles in the regulation of flavonoid biosynthesis in S. baicalensis. This study provided a large number of highly accurate full-length transcripts, which represents a valuable genetic resource for further research of the molecular biology of S. baicalensis, such as the development, breeding, and biosynthesis of active ingredients.


April 21, 2020  |  

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.


April 21, 2020  |  

Chromosomal-level assembly of the blolsod clam, Scapharca (Anadara) broughtonii, using long sequence reads and Hi-C.

The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the family Arcidae. Efforts to study their population genetics, breeding, cultivation, and stock enrichment have been somewhat hindered by the lack of a reference genome. Herein, we report the complete genome sequence of S. broughtonii, a first reference genome of the family Arcidae.A total of 75.79 Gb clean data were generated with the Pacific Biosciences and Oxford Nanopore platforms, which represented approximately 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5-Mb genome, with a contig N50 of 1.80 Mb and scaffold N50 of 45.00 Mb. Genome Hi-C scaffolding resulted in 19 chromosomes containing 99.35% of bases in the assembled genome. Genome annotation revealed that nearly half of the genome (46.1%) is composed of repeated sequences, while 24,045 protein-coding genes were predicted and 84.7% of them were annotated.We report here a chromosomal-level assembly of the S. broughtonii genome based on long-read sequencing and Hi-C scaffolding. The genomic data can serve as a reference for the family Arcidae and will provide a valuable resource for the scientific community and aquaculture sector. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

De novo genome assembly of the endangered Acer yangbiense, a plant species with extremely small populations endemic to Yunnan Province, China.

Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ~666 Mb, with 13 chromosomes covering ~97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization.Here, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing.

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.


April 21, 2020  |  

The genome assembly and annotation of yellowhorn (Xanthoceras sorbifolium Bunge).

Yellowhorn (Xanthoceras sorbifolium Bunge), a deciduous shrub or small tree native to north China, is of great economic value. Seeds of yellowhorn are rich in oil containing unsaturated long-chain fatty acids that have been used for producing edible oil and nervonic acid capsules. However, the lack of a high-quality genome sequence hampers the understanding of its evolution and gene functions.In this study, a whole genome of yellowhorn was sequenced and assembled by integration of Illumina sequencing, Pacific Biosciences single-molecule real-time sequencing, 10X Genomics linked reads, Bionano optical maps, and Hi-C. The yellowhorn genome assembly was 439.97 Mb, which comprised 15 pseudo-chromosomes covering 95.42% (419.84 Mb) of the assembled genome. The repetitive fractions accounted for 56.39% of the yellowhorn genome. The genome contained 21,059 protein-coding genes. Of them, 18,503 (87.86%) genes were found to be functionally annotated with =1 “annotation” term by searching against other databases. Transcriptomic analysis showed that 341, 135, 125, 113, and 100 genes were specifically expressed in hermaphrodite flower, staminate flower, young fruit, leaf, and shoot, respectively. Phylogenetic analysis suggested that yellowhorn and Dimocarpus longan diverged from their most recent common ancestor ~46 million years ago.The availability and subsequent annotation of the yellowhorn genome, as well as the identification of tissue-specific functional genes, provides a valuable reference for plant comparative genomics, evolutionary studies, and molecular design breeding. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Pseudomolecule-level assembly of the Chinese oil tree yellowhorn (Xanthoceras sorbifolium) genome.

Yellowhorn (Xanthoceras sorbifolium) is a species of the Sapindaceae family native to China and is an oil tree that can withstand cold and drought conditions. A pseudomolecule-level genome assembly for this species will not only contribute to understanding the evolution of its genes and chromosomes but also bring yellowhorn breeding into the genomic era.Here, we generated 15 pseudomolecules of yellowhorn chromosomes, on which 97.04% of scaffolds were anchored, using the combined Illumina HiSeq, Pacific Biosciences Sequel, and Hi-C technologies. The length of the final yellowhorn genome assembly was 504.2 Mb with a contig N50 size of 1.04 Mb and a scaffold N50 size of 32.17 Mb. Genome annotation revealed that 68.67% of the yellowhorn genome was composed of repetitive elements. Gene modelling predicted 24,672 protein-coding genes. By comparing orthologous genes, the divergence time of yellowhorn and its close sister species longan (Dimocarpus longan) was estimated at ~33.07 million years ago. Gene cluster and chromosome synteny analysis demonstrated that the yellowhorn genome shared a conserved genome structure with its ancestor in some chromosomes.This genome assembly represents a high-quality reference genome for yellowhorn. Integrated genome annotations provide a valuable dataset for genetic and molecular research in this species. We did not detect whole-genome duplication in the genome. The yellowhorn genome carries syntenic blocks from ancient chromosomes. These data sources will enable this genome to serve as an initial platform for breeding better yellowhorn cultivars. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Still Something to Discover: Novel Insights into Escherichia coli Phage Diversity and Taxonomy.

The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.


April 21, 2020  |  

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and B vitamins. Further analyses of reduced-coverage genome sequences of 16 Carya and 2 Juglans species provide additional phylogenetic perspective on crop wild relatives.Cooperative characterization of these valuable resources provides a window to their evolutionary development and a valuable foundation for future crop improvement. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.


April 21, 2020  |  

Complete chloroplast genome sequences of Kaempferia galanga and Kaempferia elegans: Molecular structures and comparative analysis.

Kaempferia galanga and Kaempferia elegans, which belong to the genus Kaempferia family Zingiberaceae, are used as valuable herbal medicine and ornamental plants, respectively. The chloroplast genomes have been used for molecular markers, species identification and phylogenetic studies. In this study, the complete chloroplast genome sequences of K. galanga and K. elegans are reported. Results show that the complete chloroplast genome of K. galanga is 163,811 bp long, having a quadripartite structure with large single copy (LSC) of 88,405 bp and a small single copy (SSC) of 15,812 bp separated by inverted repeats (IRs) of 29,797 bp. Similarly, the complete chloroplast genome of K. elegans is 163,555 bp long, having a quadripartite structure in which IRs of 29,773 bp length separates 88,020 bp of LSC and 15,989 bp of SSC. A total of 111 genes in K. galanga and 113 genes in K. elegans comprised 79 protein-coding genes and 4 ribosomal RNA (rRNA) genes, as well as 28 and 30 transfer RNA (tRNA) genes in K. galanga and K. elegans, respectively. The gene order, GC content and orientation of the two Kaempferia chloroplast genomes exhibited high similarity. The location and distribution of simple sequence repeats (SSRs) and long repeat sequences were determined. Eight highly variable regions between the two Kaempferia species were identified and 643 mutation events, including 536 single-nucleotide polymorphisms (SNPs) and 107 insertion/deletions (indels), were accurately located. Sequence divergences of the whole chloroplast genomes were calculated among related Zingiberaceae species. The phylogenetic analysis based on SNPs among eleven species strongly supported that K. galanga and K. elegans formed a cluster within Zingiberaceae. This study identified the unique characteristics of the entire K. galanga and K. elegans chloroplast genomes that contribute to our understanding of the chloroplast DNA evolution within Zingiberaceae species. It provides valuable information for phylogenetic analysis and species identification within genus Kaempferia.


April 21, 2020  |  

The Modern View of B Chromosomes Under the Impact of High Scale Omics Analyses.

Supernumerary B chromosomes (Bs) are extra karyotype units in addition to A chromosomes, and are found in some fungi and thousands of animals and plant species. Bs are uniquely characterized due to their non-Mendelian inheritance, and represent one of the best examples of genomic conflict. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. A classical concept based on cytogenetics and genetics is that Bs are selfish and abundant with DNA repeats and transposons, and in most cases, they do not carry any function. However, recently, the modern quantum development of high scale multi-omics techniques has shifted B research towards a new-born field that we call “B-omics”. We review the recent literature and add novel perspectives to the B research, discussing the role of new technologies to understand the mechanistic perspectives of the molecular evolution and function of Bs. The modern view states that B chromosomes are enriched with genes for many significant biological functions, including but not limited to the interesting set of genes related to cell cycle and chromosome structure. Furthermore, the presence of B chromosomes could favor genomic rearrangements and influence the nuclear environment affecting the function of other chromatin regions. We hypothesize that B chromosomes might play a key function in driving their transmission and maintenance inside the cell, as well as offer an extra genomic compartment for evolution.


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