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September 22, 2019

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

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September 22, 2019

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

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September 22, 2019

High-resolution community profiling of arbuscular mycorrhizal fungi.

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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September 22, 2019

Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications.

Moso bamboo (Phyllostachys edulis) is a well-known bamboo species of high economic value in the textile industry due to its rapid growth. Phytohormones, which are master regulators of growth and development, serve as important endogenous signals. However, the mechanisms through which phytohormones regulate growth in moso bamboo remain unknown to date.Here, we reported that exogenous gibberellins (GA) applications resulted in a significantly increased internode length and lignin condensation. Transcriptome sequencing revealed that photosynthesis-related genes were enriched in the GA-repressed gene class, which was consistent with the decrease in leaf chlorophyll concentrations and the lower rate of photosynthesis following GA treatment. Exogenous GA applications on seedlings are relatively easy to perform, thus we used 4-week-old whole seedlings of bamboo for GA- treatment followed by high throughput sequencing. In this study, we identified 932 cis-nature antisense transcripts (cis-NATs), and 22,196 alternative splicing (AS) events in total. Among them, 42 cis-nature antisense transcripts (cis-NATs) and 442 AS events were differentially expressed upon exposure to exogenous GA3, suggesting that post-transcriptional regulation might be also involved in the GA3 response. Targets of differential expression of cis-NATs included genes involved in hormone receptor, photosynthesis and cell wall biogenesis. For example, LAC4 and its corresponding cis-NATs were GA3-induced, and may be involved in the accumulation of lignin, thus affecting cell wall composition.This study provides novel insights illustrating how GA alters post-transcriptional regulation and will shed light on the underlying mechanism of growth modulated by GA in moso bamboo.

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September 22, 2019

Analysis of transcripts and splice isoforms in red clover (Trifolium pratense L.) by single-molecule long-read sequencing.

Red clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover.In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2194 novel isoforms from novel genes were identified. A total of 5492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8719 including 186,517 transcripts have at least one poly(A) site. Furthermore, we identified 4333 long non-coding RNAs and 3762 fusion transcripts.We analyzed full-length transcriptome of red clover with PacBio SMRT. Those new findings provided important information for improving red clover draft genome annotation and fully characterization of red clover transcriptome.

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September 22, 2019

Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing differential intron retention, and isoform ratio switching.

Abiotic stresses affect plant physiology, development, growth, and alter pre-mRNA splicing. Western poplar is a model woody tree and a potential bioenergy feedstock. To investigate the extent of stress-regulated alternative splicing (AS), we conducted an in-depth survey of leaf, root, and stem xylem transcriptomes under drought, salt, or temperature stress. Analysis of approximately one billion of genome-aligned RNA-Seq reads from tissue- or stress-specific libraries revealed over fifteen millions of novel splice junctions. Transcript models supported by both RNA-Seq and single molecule isoform sequencing (Iso-Seq) data revealed a broad array of novel stress- and/or tissue-specific isoforms. Analysis of Iso-Seq data also resulted in the discovery of 15,087 novel transcribed regions of which 164 show AS. Our findings demonstrate that abiotic stresses profoundly perturb transcript isoform profiles and trigger widespread intron retention (IR) events. Stress treatments often increased or decreased retention of specific introns – a phenomenon described here as differential intron retention (DIR). Many differentially retained introns were regulated in a stress- and/or tissue-specific manner. A subset of transcripts harboring super stress-responsive DIR events showed persisting fluctuations in the degree of IR across all treatments and tissue types. To investigate coordinated dynamics of intron-containing transcripts in the study we quantified absolute copy number of isoforms of two conserved transcription factors (TFs) using Droplet Digital PCR. This case study suggests that stress treatments can be associated with coordinated switches in relative ratios between fully spliced and intron-retaining isoforms and may play a role in adjusting transcriptome to abiotic stresses.

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September 22, 2019

Full-length transcriptome sequences and the identification of putative genes for flavonoid biosynthesis in safflower.

The flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study.High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1-2, 2-3 and 3-6 k) library construction. Five cells were carried (2 cells for 1-2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study.Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

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September 22, 2019

Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research.

The large and complex hexaploid genome has greatly hindered genomics studies of common wheat (Triticum aestivum, AABBDD). Here, we investigated transcripts in common wheat developing caryopses using the emerging single-molecule real-time (SMRT) sequencing technology PacBio RSII, and assessed the resultant data for improving common wheat genome annotation and grain transcriptome research.We obtained 197,709 full-length non-chimeric (FLNC) reads, 74.6 % of which were estimated to carry complete open reading frame. A total of 91,881 high-quality FLNC reads were identified and mapped to 16,188 chromosomal loci, corresponding to 13,162 known genes and 3026 new genes not annotated previously. Although some FLNC reads could not be unambiguously mapped to the current draft genome sequence, many of them are likely useful for studying highly similar homoeologous or paralogous loci or for improving chromosomal contig assembly in further research. The 91,881 high-quality FLNC reads represented 22,768 unique transcripts, 9591 of which were newly discovered. We found 180 transcripts each spanning two or three previously annotated adjacent loci, suggesting that they should be merged to form correct gene models. Finally, our data facilitated the identification of 6030 genes differentially regulated during caryopsis development, and full-length transcripts for 72 transcribed gluten gene members that are important for the end-use quality control of common wheat.Our work demonstrated the value of PacBio transcript sequencing for improving common wheat genome annotation through uncovering the loci and full-length transcripts not discovered previously. The resource obtained may aid further structural genomics and grain transcriptome studies of common wheat.

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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

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

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

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

Soil bacterial communities are shaped by temporal and environmental filtering: evidence from a long-term chronosequence.

Soil microbial communities are abundant, hyper-diverse and mediate global biogeochemical cycles, but we do not yet understand the processes mediating their assembly. Current hypothetical frameworks suggest temporal (e.g. dispersal limitation) and environmental (e.g. soil pH) filters shape microbial community composition; however, there is limited empirical evidence supporting this framework in the hyper-diverse soil environment, particularly at large spatial (i.e. regional to continental) and temporal (i.e. 100 to 1000 years) scales. Here, we present evidence from a long-term chronosequence (4000 years) that temporal and environmental filters do indeed shape soil bacterial community composition. Furthermore, nearly 20 years of environmental monitoring allowed us to control for potentially confounding environmental variation. Soil bacterial communities were phylogenetically distinct across the chronosequence. We determined that temporal and environmental factors accounted for significant portions of bacterial phylogenetic structure using distance-based linear models. Environmental factors together accounted for the majority of phylogenetic structure, namely, soil temperature (19%), pH (17%) and litter carbon:nitrogen (C:N; 17%). However, of all individual factors, time since deglaciation accounted for the greatest proportion of bacterial phylogenetic structure (20%). Taken together, our results provide empirical evidence that temporal and environmental filters act together to structure soil bacterial communities across large spatial and long-term temporal scales. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.

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September 22, 2019

The small peptide world in long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.

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