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September 22, 2019

Transcriptome profiling using Illumina- and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection.

Hot pepper (Capsicum annuum L.) is becoming an increasingly important vegetable crop in the world. Cucumber mosaic virus (CMV) is a destructive virus that can cause leaf distortion and fruit lesions, affecting pepper production. However, studies on the response to CMV infection in pepper at the transcriptional level are limited. In this study, the transcript profiles of pepper leaves after CMV infection were investigated using Illumina and single-molecule real-time (SMRT) RNA-sequencing (RNA-seq). A total of 2143 differentially expressed genes (DEGs) were identified at five different stages. Gene ontology (GO) and KEGG analysis revealed that these DEGs were involved in the response to stress, defense response and plant-pathogen interaction pathways. Among these DEGs, several key genes that consistently appeared in studies of plant-pathogen interactions had increased transcript abundance after inoculation, including chitinase, pathogenesis-related (PR) protein, TMV resistance protein, WRKY transcription factor and jasmonate ZIM-domain protein. Four of these DEGs were further validated by quantitative real-time RT-PCR (qRT-PCR). Furthermore, a total of 73, 597 alternative splicing (AS) events were identified in the pepper leaves after CMV infection, distributed in 12, 615 genes. The intron retention of WRKY33 (Capana09g001251) might be involved in the regulation of CMV infection. Taken together, our study provides a transcriptome-wide insight into the molecular basis of resistance to CMV infection in pepper leaves and potential candidate genes for improving resistance cultivars. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

De novo transcriptome assembly of the Chinese pearl barley, adlay, by full-length isoform and short-read RNA sequencing.

Adlay (Coix lacryma-jobi) is a tropical grass that has long been used in traditional Chinese medicine and is known for its nutritional benefits. Recent studies have shown that vitamin E compounds in adlay protect against chronic diseases such as cancer and heart disease. However, the molecular basis of adlay’s health benefits remains unknown. Here, we generated adlay gene sets by de novo transcriptome assembly using long-read isoform sequencing (Iso-Seq) and short-read RNA-Sequencing (RNA-Seq). The gene sets obtained from Iso-seq and RNA-seq contained 31,177 genes and 57,901 genes, respectively. We confirmed the validity of the assembled gene sets by experimentally analyzing the levels of prolamin and vitamin E biosynthesis-associated proteins in adlay plant tissues and seeds. We compared the screened adlay genes with known gene families from closely related plant species, such as rice, sorghum and maize. We also identified tissue-specific genes from the adlay leaf, root, and young and mature seed, and experimentally validated the differential expression of 12 randomly-selected genes. Our study of the adlay transcriptome will provide a valuable resource for genetic studies that can enhance adlay breeding programs in the future.

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September 22, 2019

Transcriptome sequencing and comparative analysis of differentially-expressed isoforms in the roots of Halogeton glomeratus under salt stress.

Although Halogeton glomeratus (H. glomeratus) has been confirmed to have a unique mechanism to regulate Na+efflux from the cytoplasm and compartmentalize Na+into leaf vacuoles, little is known about the salt tolerance mechanisms of roots under salinity stress. In the present study, transcripts were sequenced using the BGISEQ-500 sequencing platform (BGI, Wuhan, China). After quality control, approximately 24.08 million clean reads were obtained and the average mapping ratio to the reference gene was 70.00%. When comparing salt-treated samples with the control, a total of 550, 590, 1411 and 2063 DEIs were identified at 2, 6, 24 and 72h, respectively. Numerous differentially-expressed isoforms that play important roles in response and adaptation to salt condition are related to metabolic processes, cellular processes, single-organism processes, localization, biological regulation, responses to stimulus, binding, catalytic activity and transporter activity. Fifty-eight salt-induced isoforms were common to different stages of salt stress; most of these DEIs were related to signal transduction and transporters, which maybe the core isoforms regulating Na+uptake and transport in the roots of H. glomeratus. The expression patterns of 18 DEIs that were detected by quantitative real-time polymerase chain reaction were consistent with their respective changes in transcript abundance as identified by RNA-Seq technology. The present study thoroughly explored potential isoforms involved in salt tolerance on H. glomeratus roots at five time points. Our results may serve as an important resource for the H. glomeratus research community, improving our understanding of salt tolerance in halophyte survival under high salinity stress. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Comparative genomic analysis of Sulfurospirillum cavolei MES reconstructed from the metagenome of an electrosynthetic microbiome.

Sulfurospirillum spp. play an important role in sulfur and nitrogen cycling, and contain metabolic versatility that enables reduction of a wide range of electron acceptors, including thiosulfate, tetrathionate, polysulfide, nitrate, and nitrite. Here we describe the assembly of a Sulfurospirillum genome obtained from the metagenome of an electrosynthetic microbiome. The ubiquity and persistence of this organism in microbial electrosynthesis systems suggest it plays an important role in reactor stability and performance. Understanding why this organism is present and elucidating its genetic repertoire provide a genomic and ecological foundation for future studies where Sulfurospirillum are found, especially in electrode-associated communities. Metabolic comparisons and in-depth analysis of unique genes revealed potential ecological niche-specific capabilities within the Sulfurospirillum genus. The functional similarities common to all genomes, i.e., core genome, and unique gene clusters found only in a single genome were identified. Based upon 16S rRNA gene phylogenetic analysis and average nucleotide identity, the Sulfurospirillum draft genome was found to be most closely related to Sulfurospirillum cavolei. Characterization of the draft genome described herein provides pathway-specific details of the metabolic significance of the newly described Sulfurospirillum cavolei MES and, importantly, yields insight to the ecology of the genus as a whole. Comparison of eleven sequenced Sulfurospirillum genomes revealed a total of 6246 gene clusters in the pan-genome. Of the total gene clusters, 18.5% were shared among all eleven genomes and 50% were unique to a single genome. While most Sulfurospirillum spp. reduce nitrate to ammonium, five of the eleven Sulfurospirillum strains encode for a nitrous oxide reductase (nos) cluster with an atypical nitrous-oxide reductase, suggesting a utility for this genus in reduction of the nitrous oxide, and as a potential sink for this potent greenhouse gas.

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September 22, 2019

De novo transcriptome assembly of drought tolerant CAM plants, Agave deserti and Agave tequilana.

Agaves are succulent monocotyledonous plants native to xeric environments of North America. Because of their adaptations to their environment, including crassulacean acid metabolism (CAM, a water-efficient form of photosynthesis), and existing technologies for ethanol production, agaves have gained attention both as potential lignocellulosic bioenergy feedstocks and models for exploring plant responses to abiotic stress. However, the lack of comprehensive Agave sequence datasets limits the scope of investigations into the molecular-genetic basis of Agave traits.Here, we present comprehensive, high quality de novo transcriptome assemblies of two Agave species, A. tequilana and A. deserti, built from short-read RNA-seq data. Our analyses support completeness and accuracy of the de novo transcriptome assemblies, with each species having a minimum of approximately 35,000 protein-coding genes. Comparison of agave proteomes to those of additional plant species identifies biological functions of gene families displaying sequence divergence in agave species. Additionally, a focus on the transcriptomics of the A. deserti juvenile leaf confirms evolutionary conservation of monocotyledonous leaf physiology and development along the proximal-distal axis.Our work presents a comprehensive transcriptome resource for two Agave species and provides insight into their biology and physiology. These resources are a foundation for further investigation of agave biology and their improvement for bioenergy development.

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September 22, 2019

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes.

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 a-, 11 ?-, one d- and five ?-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of a-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The d-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic a-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

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September 22, 2019

Global identification of the full-length transcripts and alternative splicing related to phenolic acid biosynthetic genes in Salvia miltiorrhiza.

Salvianolic acids are among the main bioactive components in Salvia miltiorrhiza, and their biosynthesis has attracted widespread interest. However, previous studies on the biosynthesis of phenolic acids using next-generation sequencing platforms are limited with regard to the assembly of full-length transcripts. Based on hybrid-seq (next-generation and single molecular real-time sequencing) of the S. miltiorrhiza root transcriptome, we experimentally identified 15 full-length transcripts and four alternative splicing events of enzyme-coding genes involved in the biosynthesis of rosmarinic acid. Moreover, we herein demonstrate that lithospermic acid B accumulates in the phloem and xylem of roots, in agreement with the expression patterns of the identified key genes related to rosmarinic acid biosynthesis. According to co-expression patterns, we predicted that six candidate cytochrome P450s and five candidate laccases participate in the salvianolic acid pathway. Our results provide a valuable resource for further investigation into the synthetic biology of phenolic acids in S. miltiorrhiza.

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September 22, 2019

Complete genome sequence of Endomicrobium proavitum, a free-living relative of the intracellular symbionts of termite gut flagellates (phylum Elusimicrobia).

We sequenced the complete genome of Endomicrobium proavitum strain Rsa215, the first isolate of the class Endomicrobia (phylum Elusimicrobia). It is the closest free-living relative of the endosymbionts of termite gut flagellates and thereby provides an excellent model for studying the evolutionary processes during the establishment of an intracellular symbiosis. Copyright © 2015 Zheng and Brune.

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September 22, 2019

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass.

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

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September 22, 2019

Multiple regulatory networks are activated during cold stress in Medicago sativa L.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.

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September 22, 2019

The genomic and functional landscapes of developmental plasticity in the American cockroach.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its adaptation to urban environments and developmental plasticity. In comparison with other insects, expansions of gene families in P. americana exist for most core gene families likely associated with environmental adaptation, such as chemoreception and detoxification. Multiple pathways regulating metamorphic development are well conserved, and RNAi experiments inform on key roles of 20-hydroxyecdysone, juvenile hormone, insulin, and decapentaplegic signals in regulating plasticity. Our analyses reveal a high level of sequence identity in genes between the American cockroach and two termite species, advancing it as a valuable model to study the evolutionary relationships between cockroaches and termites.

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September 22, 2019

Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids, derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding a total of 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

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September 22, 2019

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

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September 22, 2019

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

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September 22, 2019

Generation and comparative analysis of full-length transcriptomes in sweetpotato and its putative wild ancestor I. trifida.

Sweetpotato [Ipomoea batatas (L.) Lam.] is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither high-quality reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid are still lacking, which in turn impedes progress in research studies in sweetpotato functional genomics and molecular breeding. In this study, we apply a combination of second- and third-generation sequencing technologies to sequence full-length transcriptomes in sweetpotato and its putative ancestor I. trifida. In total, we obtained 53,861/51,184 high-quality transcripts, which includes 34,963/33,637 putative full-length cDNA sequences, from sweetpotato/I. trifida. Amongst, we identified 104,540/94,174 open reading frames, 1476/1475 transcription factors, 25,315/27,090 simple sequence repeats, 417/531 long non-coding RNAs out of the sweetpotato/I. trifida dataset. By utilizing public available genomic contigs, we analyzed the gene features (including exon number, exon size, intron number, intron size, exon-intron structure) of 33,119 and 32,793 full-length transcripts in sweetpotato and I. trifida, respectively. Furthermore, comparative analysis between our transcript datasets and other large-scale cDNA datasets from different plant species enables us assessing the quality of public datasets, estimating the genetic similarity across relative species, and surveyed the evolutionary pattern of genes. Overall, our study provided fundamental resources of large-scale full-length transcripts in sweetpotato and its putative ancestor, for the first time, and would facilitate structural, functional and comparative genomics studies in this important crop.

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