scholarly journals Long-read sequencing of Chrysanthemum morifolium cv. ‘Hangju’ transcriptome reveals flavonoid biosynthesis and regulation

2019 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Chrysanthemum Morifolium ◽  
Flavonoid Biosynthesis ◽  
Full Length ◽  
Transcriptome Data ◽  
Bioactive Components ◽  
Smrt Sequencing ◽  
Major Genes ◽  
Poor Understanding ◽  
Long Read

Abstract Abstract Background : The capitulum of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China for antioxidant and anti-inflammatory. Flavonoids as one of the bioactive components in C . morifolium have a poor understanding in their biosynthesis and regulation. Nowadays, transcriptome sequencing as an effective method was used in capturing the transcripts information. So, single-molecule real-time (SMRT) sequencing was performed to obtain the full length of genes involved in flavonoid biosynthesis and regulation in C . morifolium . Results : The high-quality RNA was extracted from the capitulum of C . morifolium at different development stages, and it was constructed into two libraries (0-5 kb and 4.5-10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with mean length of 2,009 bp were captured. Of which, 2,083 transcripts were annotated in the pathway related to the flavonoid biosynthesis and 56 isoforms were annotated as CHS , CHI , F3H , F3’H , FNS Ⅱ , FLS , DFR and ANS genes. Based on the gene expression level at different stages, we predicted the major genes involved in the flavonoid biosynthesis. And we found two candidate MYB factors (CmMYBF1 and CmMYBF2) activating the flavonol biosynthesis by phylogenetic analysis. Conclusions : Based on the full-length transcriptome data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C . morifolium were predicted in our study. The results provide a valuable theoretical basis for introduction and cultivation of C. morifolium cv. ‘Hangju’.

scholarly journals Long-read sequencing of Chrysanthemum morifolium cv. ‘Hangju’ transcriptome reveals flavonoid biosynthesis and regulation

2020 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Chrysanthemum Morifolium ◽  
Flavonoid Biosynthesis ◽  
Full Length ◽  
Bioactive Components ◽  
Smrt Sequencing ◽  
Major Genes ◽  
Long Read ◽  
Gene Expression Levels

Abstract Background: The inflorescence of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China due to its antioxidant and anti-inflammatory properties. The biosynthesis and regulation of flavonoids, a group of bioactive components, in C. morifolium are poorly understood. Transcriptome sequencing is an effective method for obtaining transcript information. Therefore, single-molecule real-time (SMRT) sequencing was performed to obtain the full-length genes involved in flavonoid biosynthesis and regulation in C. morifolium.Results: High-quality RNA was extracted from the inflorescence of C. morifolium at different developmental stages and used to construct two libraries (0-5 kb and 4.5-10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with a mean length of 2,009 bp were obtained. Of these, 2,083 transcripts were annotated to pathways related to flavonoid biosynthesis, and 56 isoforms were annotated as CHS, CHI, F3H, F3’H, FNS Ⅱ, FLS, DFR and ANS genes. Based on gene expression levels at different stages, we predicted the major genes involved in flavonoid biosynthesis. By phylogenetic analysis, we found two candidate MYB transcription factors (CmMYBF1 and CmMYBF2) activating flavonol biosynthesis.Conclusions: Based on the full-length transcriptomic data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C. morifolium were predicted in our study. The results provide a valuable theoretical basis for the introduction and cultivation of C. morifolium cv. ‘Hangju’.

scholarly journals Long-read sequencing of Chrysanthemum morifolium cv. ‘Hangju’ transcriptome reveals flavonoid biosynthesis and regulation

2019 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Chrysanthemum Morifolium ◽  
Flavonoid Biosynthesis ◽  
Full Length ◽  
Bioactive Components ◽  
Smrt Sequencing ◽  
Major Genes ◽  
Long Read ◽  
Gene Expression Levels

Abstract Background: The inflorescence of Chrysanthemum morifolium cv. ‘Hangju’ has been widely used in China due to its antioxidant and anti-inflammatory properties. The biosynthesis and regulation of flavonoids, a group of bioactive components, in C. morifolium are poorly understood. Transcriptome sequencing is an effective method for obtaining transcript information. Therefore, single-molecule real-time (SMRT) sequencing was performed to obtain the full-length genes involved in flavonoid biosynthesis and regulation in C. morifolium. Results: High-quality RNA was extracted from the inflorescence of C. morifolium at different developmental stages and used to construct two libraries (0-5 kb and 4.5-10 kb) for sequencing. Finally, 125,532 non-redundant isoforms with a mean length of 2,009 bp were obtained. Of these, 2,083 transcripts were annotated to pathways related to flavonoid biosynthesis, and 56 isoforms were annotated as CHS, CHI, F3H, F3’H, FNS Ⅱ, FLS, DFR and ANS genes. Based on gene expression levels at different stages, we predicted the major genes involved in flavonoid biosynthesis. By phylogenetic analysis, we found two candidate MYB transcription factors (CmMYBF1 and CmMYBF2) activating flavonol biosynthesis. Conclusions: Based on the full-length transcriptomic data and further quantitative analysis, the major genes involved in flavonoid biosynthesis and regulation in C. morifolium were predicted in our study. The results provide a valuable theoretical basis for the introduction and cultivation of C. morifolium cv. ‘Hangju’.

scholarly journals SMRT sequencing of the full-length transcriptome of the white-backed planthopper Sogatella furcifera

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9320
Author(s):  
Jing Chen ◽  
Yaya Yu ◽  
Kui Kang ◽  
Daowei Zhang
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Virus Transmission ◽  
Full Length ◽  
Transcriptome Data ◽  
Smrt Sequencing ◽  
Sogatella Furcifera ◽  
Consensus Sequences ◽  
Host Interactions ◽  
Ssr Analysis

The white-backed planthopper Sogatella furcifera is an economically important rice pest distributed throughout Asia. It damages rice crops by sucking phloem sap, resulting in stunted growth and plant virus transmission. We aimed to obtain the full-length transcriptome data of S. furcifera using PacBio single-molecule real-time (SMRT) sequencing. Total RNA extracted from S. furcifera at various developmental stages (egg, larval, and adult stages) was mixed and used to generate a full-length transcriptome for SMRT sequencing. Long non-coding RNA (lncRNA) identification, full-length coding sequence prediction, full-length non-chimeric (FLNC) read detection, simple sequence repeat (SSR) analysis, transcription factor detection, and transcript functional annotation were performed. A total of 12,514,449 subreads (15.64 Gbp, clean reads) were generated, including 630,447 circular consensus sequences and 388,348 FLNC reads. Transcript cluster analysis of the FLNC reads revealed 251,109 consensus reads including 29,700 high-quality reads. Additionally, 100,360 SSRs and 121,395 coding sequences were identified using SSR analysis and ANGEL software, respectively. Furthermore, 44,324 lncRNAs were annotated using four tools and 1,288 transcription factors were identified. In total, 95,495 transcripts were functionally annotated based on searches of seven different databases. To the best of our knowledge, this is the first study of the full-length transcriptome of the white-backed planthopper obtained using SMRT sequencing. The acquired transcriptome data can facilitate further studies on the ecological and viral-host interactions of this agricultural pest.

scholarly journals PacBio single-molecule long-read sequencing shed new light on the complexity of the Carex breviculmis transcriptome

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ke Teng ◽  
Wenjun Teng ◽  
Haifeng Wen ◽  
Yuesen Yue ◽  
Weier Guo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Average Length ◽  
Transcriptome Profiling ◽  
Full Length ◽  
Regulation Mechanism ◽  
Forage Production ◽  
Smrt Sequencing ◽  
Reference Transcriptome ◽  
Long Read ◽  
Ecological Conservation

Abstract Background Carex L., a grass genus commonly known as sedges, is distributed worldwide and contributes constructively to turf management, forage production, and ecological conservation. The development of next-generation sequencing (NGS) technologies has considerably improved our understanding of transcriptome complexity of Carex L. and provided a valuable genetic reference. However, the current transcriptome is not satisfactory mainly because of the enormous difficulty in obtaining full-length transcripts. Results In this study, we employed PacBio single-molecule long-read sequencing (SMRT) technology for whole-transcriptome profiling in Carex breviculmis. We generated 60,353 high-confidence non-redundant transcripts with an average length of 2302-bp. A total of 3588 alternative splicing events, and 1273 long non-coding RNAs were identified. Furthermore, 40,347 complete coding sequences were predicted, providing an informative reference transcriptome. In addition, the transcriptional regulation mechanism of C. breviculmis in response to shade stress was further explored by mapping the NGS data to the reference transcriptome constructed by SMRT sequencing. Conclusions This study provided a full-length reference transcriptome of C. breviculmis using the SMRT sequencing method for the first time. The transcriptome atlas obtained will not only facilitate future functional genomics studies but also pave the way for further selective and genic engineering breeding projects for C. breviculmis.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Molecule ◽  
High Fat Diet ◽  
Human Heart ◽  
Full Length ◽  
Funding Source ◽  
Smrt Sequencing ◽  
High Fat ◽  
Exon 1 ◽  
Novel Exon

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

scholarly journals PacBio Single-Molecule Long-Read Sequencing Reveals Genes Tolerating Manganese Stress in Schima superba Saplings

2021 ◽  
Vol 12 ◽  
Author(s):  
Fiza Liaquat ◽  
Muhammad Farooq Hussain Munis ◽  
Samiah Arif ◽  
Urooj Haroon ◽  
Jianxin Shi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gene Annotation ◽  
Treated Group ◽  
Full Length ◽  
Open Reading Frames ◽  
Interacting Protein ◽  
Schima Superba ◽  
Long Read ◽  
First Time ◽  
Potential Tool

Schima superba (Theaceae) is a subtropical evergreen tree and is used widely for forest firebreaks and gardening. It is a plant that tolerates salt and typically accumulates elevated amounts of manganese in the leaves. With large ecological amplitude, this tree species grows quickly. Due to its substantial biomass, it has a great potential for soil remediation. To evaluate the thorough framework of the mRNA, we employed PacBio sequencing technology for the first time to generate S. Superba transcriptome. In this analysis, overall, 511,759 full length non-chimeric reads were acquired, and 163,834 high-quality full-length reads were obtained. Overall, 93,362 open reading frames were obtained, of which 78,255 were complete. In gene annotation analyses, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Non-Redundant (Nr) databases were allocated 91,082, 71,839, 38,914, and 38,376 transcripts, respectively. To identify long non-coding RNAs (lncRNAs), we utilized four computational methods associated with protein families (Pfam), Cooperative Data Classification (CPC), Coding Assessing Potential Tool (CPAT), and Coding Non-Coding Index (CNCI) databases and observed 8,551, 9,174, 20,720, and 18,669 lncRNAs, respectively. Moreover, nine genes were randomly selected for the expression analysis, which showed the highest expression of Gene 6 (Na_Ca_ex gene), and CAX (CAX-interacting protein 4) was higher in manganese (Mn)-treated group. This work provided significant number of full-length transcripts and refined the annotation of the reference genome, which will ease advanced genetic analyses of S. superba.

scholarly journals Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet–Biedl Syndrome 9 (BBS9) deletion

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Jennifer Reiner ◽  
Laura Pisani ◽  
Wanqiong Qiao ◽  
Ram Singh ◽  
Yao Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
Bardet Biedl Syndrome ◽  
Long Read

scholarly journals Single-Molecule Long-Read Sequencing Reveals the Diversity of Full-Length Transcripts in Leaves of Gnetum (Gnetales)

2019 ◽  
Vol 20 (24) ◽  
pp. 6350 ◽  
Author(s):  
Nan Deng ◽  
Chen Hou ◽  
Fengfeng Ma ◽  
Caixia Liu ◽  
Yuxin Tian
Keyword(s):  
Single Molecule ◽  
Developmental Stages ◽  
Alternative Polyadenylation ◽  
Full Length ◽  
Stomatal Development ◽  
Rna Seq ◽  
Leaf Transcriptome ◽  
Long Read ◽  
Non Coding Rnas ◽  
A Site

The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.

scholarly journals Single-Molecule Real-Time Sequencing of the Madhuca pasquieri (Dubard) Lam. Transcriptome Reveals the Diversity of Full-Length Transcripts

Forests ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 866
Author(s):  
Lei Kan ◽  
Qicong Liao ◽  
Zhiyao Su ◽  
Yushan Tan ◽  
Shuyu Wang ◽  
...  
Keyword(s):  
Seed Germination ◽  
Single Molecule ◽  
Developmental Stages ◽  
De Novo ◽  
Full Length ◽  
Wild Plant ◽  
Transcript Isoforms ◽  
Long Read ◽  
Full Length Transcript ◽  
Generation Sequencing

Madhuca pasquieri (Dubard) Lam. is a tree on the International Union for Conservation of Nature Red List and a national key protected wild plant (II) of China, known for its seed oil and timber. However, lacking of genomic and transcriptome data for this species hampers study of its reproduction, utilization, and conservation. Here, single-molecule long-read sequencing (PacBio) and next-generation sequencing (Illumina) were combined to obtain the transcriptome from five developmental stages of M. pasquieri. Overall, 25,339 transcript isoforms were detected by PacBio, including 24,492 coding sequences (CDSs), 9440 simple sequence repeats (SSRs), 149 long non-coding RNAs (lncRNAs), and 182 alternative splicing (AS) events, a majority was retained intron (RI). A further 1058 transcripts were identified as transcriptional factors (TFs) from 51 TF families. PacBio recovered more full-length transcript isoforms with a longer length, and a higher expression level, whereas larger number of transcripts (124,405) was captured in de novo from Illumina. Using Nr, Swissprot, KOG, and KEGG databases, 24,405 transcripts (96.31%) were annotated by PacBio. Functional annotation revealed a role for the auxin, abscisic acid, gibberellin, and cytokinine metabolic pathways in seed germination and post-germination. These findings support further studies on seed germination mechanism and genome of M. pasquieri, and better protection of this endangered species.

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