scholarly journals Full-Length Transcriptome Analysis of Four Different Tissues of Cephalotaxus oliveri

2021 ◽  
Vol 22 (2) ◽  
pp. 787
Author(s):  
Ziqing He ◽  
Yingjuan Su ◽  
Ting Wang

Cephalotaxus oliveri is a tertiary relict conifer endemic to China, regarded as a national second-level protected plant in China. This species has experienced severe changes in temperature and precipitation in the past millions of years, adapting well to harsh environments. In view of global climate change and its endangered conditions, it is crucial to study how it responds to changes in temperature and precipitation for its conservation work. In this study, single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing were combined to generate the complete transcriptome of C. oliveri. Using the RNA-seq data to correct the SMRT sequencing data, the four tissues obtained 63,831 (root), 58,108 (stem), 33,013 (leaf) and 62,436 (male cone) full-length unigenes, with a N50 length of 2523, 3480, 3181, and 3267 bp, respectively. Additionally, 35,887, 11,306, 36,422, and 25,439 SSRs were detected for the male cone, leaf, root, and stem, respectively. The number of long non-coding RNAs predicted from the root was the largest (11,113), and the other tissues were 3408 (stem), 3193 (leaf), and 3107 (male cone), respectively. Functional annotation and enrichment analysis of tissue-specific expressed genes revealed the special roles in response to environmental stress and adaptability in the different four tissues. We also characterized the gene families and pathways related to abiotic factors. This work provides a comprehensive transcriptome resource for C. oliveri, and this resource will facilitate further studies on the functional genomics and adaptive evolution of C. oliveri.

2021 ◽  
Vol 12 ◽  
Author(s):  
Yupeng Cui ◽  
Xinqiang Gao ◽  
Jianshe Wang ◽  
Zengzhen Shang ◽  
Zhibin Zhang ◽  
...  

Artemisia argyi is an important medicinal plant widely utilized for moxibustion heat therapy in China. The terpenoid biosynthesis process in A. argyi is speculated to play a key role in conferring its medicinal value. However, the molecular mechanism underlying terpenoid biosynthesis remains unclear, in part because the reference genome of A. argyi is unavailable. Moreover, the full-length transcriptome of A. argyi has not yet been sequenced. Therefore, in this study, de novo transcriptome sequencing of A. argyi's root, stem, and leaf tissues was performed to obtain those candidate genes related to terpenoid biosynthesis, by combining the PacBio single-molecule real-time (SMRT) and Illumina sequencing NGS platforms. And more than 55.4 Gb of sequencing data and 108,846 full-length reads (non-chimeric) were generated by the Illumina and PacBio platform, respectively. Then, 53,043 consensus isoforms were clustered and used to represent 36,820 non-redundant transcripts, of which 34,839 (94.62%) were annotated in public databases. In the comparison sets of leaves vs roots, and leaves vs stems, 13,850 (7,566 up-regulated, 6,284 down-regulated) and 9,502 (5,284 up-regulated, 4,218 down-regulated) differentially expressed transcripts (DETs) were obtained, respectively. Specifically, the expression profile and KEGG functional enrichment analysis of these DETs indicated that they were significantly enriched in the biosynthesis of amino acids, carotenoids, diterpenoids and flavonoids, as well as the metabolism processes of glycine, serine and threonine. Moreover, multiple genes encoding significant enzymes or transcription factors related to diterpenoid biosynthesis were highly expressed in the A. argyi leaves. Additionally, several transcription factor families, such as RLK-Pelle_LRR-L-1 and RLK-Pelle_DLSV, were also identified. In conclusion, this study offers a valuable resource for transcriptome information, and provides a functional genomic foundation for further research on molecular mechanisms underlying the medicinal use of A. argyi leaves.


2020 ◽  
Vol 10 (10) ◽  
pp. 3505-3514
Author(s):  
Hongmei Zhuang ◽  
Qiang Wang ◽  
Hongwei Han ◽  
Huifang Liu ◽  
Hao Wang

To generate the full-length transcriptome of Xinjiang green and purple turnips, Brassica rapa var. Rapa, using single-molecule real-time (SMRT) sequencing. The samples of two varieties of Brassica rapa var. Rapa at five developmental stages were collected and combined to perform SMRT sequencing. Meanwhile, next generation sequencing was performed to correct SMRT sequencing data. A series of analyses were performed to investigate the transcript structure. Finally, the obtained transcripts were mapped to the genome of Brassica rapa ssp. pekinesis Chiifu to identify potential novel transcripts. For green turnip (F01), a total of 19.54 Gb clean data were obtained from 8 cells. The number of reads of insert (ROI) and full-length non-chimeric (FLNC) reads were 510,137 and 267,666. In addition, 82,640 consensus isoforms were obtained in the isoform sequences clustering, of which 69,480 were high-quality, and 13,160 low-quality sequences were corrected using Illumina RNA seq data. For purple turnip (F02), there were 20.41 Gb clean data, 552,829 ROIs, and 274,915 FLNC sequences. A total of 93,775 consensus isoforms were obtained, of which 78,798 were high-quality, and the 14,977 low-quality sequences were corrected. Following the removal of redundant sequences, there were 46,516 and 49,429 non-redundant transcripts for F01 and F02, respectively; 7,774 and 9,385 alternative splicing events were predicted for F01 and F02; 63,890 simple sequence repeats, 59,460 complete coding sequences, and 535 long-non coding RNAs were predicted. Moreover, 5,194 and 5,369 novel transcripts were identified by mapping to Brassica rapa ssp. pekinesis Chiifu. The obtained transcriptome data may improve turnip genome annotation and facilitate further study of the Brassica rapa var. Rapa genome and transcriptome.


2017 ◽  
Author(s):  
Rachael E. Workman ◽  
Alexander M. Myrka ◽  
Elizabeth Tseng ◽  
G. William Wong ◽  
Kenneth C. Welch ◽  
...  

AbstractHummingbirds can support their high metabolic rates exclusively by oxidizing ingested sugars, which is unsurprising given their sugar-rich nectar diet and use of energetically expensive hovering flight. However, they cannot rely on dietary sugars as a fuel during fasting periods, such as during the night, at first light, or when undertaking long-distance migratory flights, and must instead rely exclusively on onboard lipids. This metabolic flexibility is remarkable both in that the birds can switch between exclusive use of each fuel type within minutes and in that de novo lipogenesis from dietary sugar precursors is the principle way in which fat stores are built, sometimes at exceptionally high rates, such as during the few days prior to a migratory flight. The hummingbird hepatopancreas is the principle location of de novo lipogenesis and likely plays a key role in fuel selection, fuel switching, and glucose homeostasis. Yet understanding how this tissue, and the whole organism, achieves and moderates high rates of energy turnover is hampered by a fundamental lack of information regarding how genes coding for relevant enzymes differ in their sequence, expression, and regulation in these unique animals. To address this knowledge gap, 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, including classification of reads and clustering of isoforms (ICE) followed by error-correction (Arrow). With COGENT, we clustered different isoforms into gene families to generate de novo gene contigs. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. We also aligned our transcriptome against the Calypte anna genome where possible. Finally, we closely examined homology of critical lipid metabolic 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 have leveraged cutting-edge technology and a novel bioinformatics pipeline to provide a compelling first direct look at the transcriptome of this incredible organism.


2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  

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)


2020 ◽  
Author(s):  
Minzhen Yin ◽  
Shanshan Chu ◽  
Tingyu Shan ◽  
Liangping Zha ◽  
Huasheng Peng

Abstract Background: Astragalus mongholicus Bunge is an important medicinal plant and has been used in traditional Chinese medicine for a long history, which is rich in isoflavonoids and triterpenoid saponins. Although these active constituents in A. mongholicus have been discovered for a long time, the molecular genetic basis of the isoflavonoid and triterpenoid saponin biosynthesis pathways is virtually unknown due to the lack of a reference genome. The combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to analyze genes involved in the biosynthetic pathways of secondary metabolites in medicinal plants has been widely recognized.Results: In this study, NGS, SMRT sequencing, and targeted compounds were combined to investigate the association between isoflavonoids and triterpenoid saponins and gene expression in roots, stems and leaves of A. mongholicus. A total of four main isoflavonoids and four astragalosides (belong to triterpenoid saponins) were measured, and 44 differentially expressed genes (DEGs) of nine gene families, 44 DEGs of 16 gene families that encode for enzymes involved in isoflavonoid and triterpenoid saponin biosynthesis were identified, separately. Additionally, transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis were analyzed, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs and 11 bZIPs. The above transcripts exhibit different expression trends in different organs.Conclusions: Our study provides important genetic information for the essential genes of isoflavonoid and triterpenoid saponin biosynthesis in A. mongholicus, and provides a basis for developing its medicinal value.


2020 ◽  
Author(s):  
shaoshan zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong chen ◽  
Renfeng xiao ◽  
...  

Abstract Background: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a more complete and correct full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and one SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the complete and correct full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.


BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shaoshan Zhang ◽  
Qiong Liu ◽  
Chengcheng Lyu ◽  
Jinsong Chen ◽  
Renfeng Xiao ◽  
...  

Abstract Background Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. Results Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. Conclusion Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yanzhi Feng ◽  
Yang Zhao ◽  
Jiajia Zhang ◽  
Baoping Wang ◽  
Chaowei Yang ◽  
...  

AbstractPaulownia catalpifolia is an important, fast-growing timber species known for its high density, color and texture. However, few transcriptomic and genetic studies have been conducted in P. catalpifolia. In this study, single-molecule real-time sequencing technology was applied to obtain the full-length transcriptome of P. catalpifolia leaves treated with varying degrees of drought stress. The sequencing data were then used to search for microsatellites, or simple sequence repeats (SSRs). A total of 28.83 Gb data were generated, 25,969 high-quality (HQ) transcripts with an average length of 1624 bp were acquired after removing the redundant reads, and 25,602 HQ transcripts (98.59%) were annotated using public databases. Among the HQ transcripts, 16,722 intact coding sequences, 149 long non-coding RNAs and 179 alternative splicing events were predicted, respectively. A total of 7367 SSR loci were distributed throughout 6293 HQ transcripts, of which 763 complex SSRs and 6604 complete SSRs. The SSR appearance frequency was 28.37%, and the average distribution distance was 5.59 kb. Among the 6604 complete SSR loci, 1–3 nucleotide repeats were dominant, occupying 97.85% of the total SSR loci, of which mono-, di- and tri-nucleotide repeats were 44.68%, 33.86% and 19.31%, respectively. We detected 112 repeat motifs, of which A/T (42.64%), AG/CT (12.22%), GA/TC (9.63%), GAA/TTC (1.57%) and CCA/TGG (1.54%) were most common in mono-, di- and tri-nucleotide repeats, respectively. The length of the repeat SSR motifs was 10–88 bp, and 4997 (75.67%) were ≤ 20 bp. This study provides a novel full-length transcriptome reference for P. catalpifolia and will facilitate the identification of germplasm resources and breeding of new drought-resistant P. catalpifolia varieties.


2019 ◽  
Author(s):  
Tao Wang ◽  
Feng Yang ◽  
Qiaosheng Guo ◽  
Qingjun Zou ◽  
Wenyan Zhang ◽  
...  

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’.


2021 ◽  
Vol 22 (19) ◽  
pp. 10443
Author(s):  
Yong Wang ◽  
Jialei Ji ◽  
Long Tong ◽  
Zhiyuan Fang ◽  
Limei Yang ◽  
...  

Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop cultivated around the world. Previous studies of cabbage gene transcripts were primarily based on next-generation sequencing (NGS) technology which cannot provide accurate information concerning transcript assembly and structure analysis. To overcome these issues and analyze the whole cabbage transcriptome at the isoform level, PacBio RS II Single-Molecule Real-Time (SMRT) sequencing technology was used for a global survey of the full-length transcriptomes of five cabbage tissue types (root, stem, leaf, flower, and silique). A total of 77,048 isoforms, capturing 18,183 annotated genes, were discovered from the sequencing data generated through SMRT. The patterns of both alternative splicing (AS) and alternative polyadenylation (APA) were comprehensively analyzed. In total, we detected 13,468 genes which had isoforms containing APA sites and 8978 genes which underwent AS events. Moreover, 5272 long non-coding RNAs (lncRNAs) were discovered, and most exhibited tissue-specific expression. In total, 3147 transcription factors (TFs) were detected and 10 significant gene co-expression network modules were identified. In addition, we found that Fusarium wilt, black rot and clubroot infection significantly influenced AS in resistant cabbage. In summary, this study provides abundant cabbage isoform transcriptome data, which promotes reannotation of the cabbage genome, deepens our understanding of their post-transcriptional regulation mechanisms, and can be used for future functional genomic research.


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