Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

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.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

BMC Genomics ◽

10.1186/s12864-020-07195-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Shaoshan Zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong Chen ◽

Renfeng Xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Smrt Sequencing ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

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.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

10.21203/rs.3.rs-24957/v3 ◽

2020 ◽

Author(s):

shaoshan zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong chen ◽

Renfeng xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Smrt Sequencing ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

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 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 full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

10.21203/rs.3.rs-24957/v4 ◽

2020 ◽

Author(s):

shaoshan zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong chen ◽

Renfeng xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Smrt Sequencing ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

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.

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana

10.21203/rs.3.rs-24957/v2 ◽

2020 ◽

Author(s):

Shaoshan Zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong Chen ◽

Renfeng Xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Steviol Glycosides ◽

Stevia Rebaudiana Bertoni ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

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 .

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Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudian

10.21203/rs.3.rs-17892/v1 ◽

2020 ◽

Author(s):

Shaoshan Zhang ◽

Qiong Liu ◽

Chengcheng Lyu ◽

Jinsong Chen ◽

Renfeng Xiao ◽

...

Keyword(s):

Single Molecule ◽

Phylogenetic Trees ◽

Leaf Tissue ◽

Stevia Rebaudiana ◽

Full Length ◽

Enzyme Assays ◽

Steviol Glycosides ◽

Leaf Tissues ◽

Sequencing Platforms ◽

Insight Into

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. Of these potential SrUGTs, twenty-seven have complete ORFs and have not been verified to date, and enzyme assays were subsequently performed, but none of them had activity towards SGs. In addition, SrUGT (SrUGT88B1-1) could utilize UDP-glucose as a sugar donor to glycosylate isoquercetin to form three products containing one, two, and three glucoses, respectively.Conclusion: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and confirmed that four enzymes (SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2) are primarily involved in the glycosylation of steviol glucosides. Moreover, the complete and correct full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

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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

European Heart Journal ◽

10.1093/ehjci/ehaa946.3582 ◽

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)

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Full-Length Transcriptome Sequences by A Combination of Sequencing Platforms Applied to Isoflavonoid and Triterpenoid Saponin Biosynthesis of Astragalus Mongholicus Bunge

10.21203/rs.3.rs-127368/v1 ◽

2020 ◽

Author(s):

Minzhen Yin ◽

Shanshan Chu ◽

Tingyu Shan ◽

Liangping Zha ◽

Huasheng Peng

Keyword(s):

Single Molecule ◽

Molecular Genetic ◽

Gene Families ◽

Triterpenoid Saponin ◽

Smrt Sequencing ◽

Triterpenoid Saponins ◽

Saponin Biosynthesis ◽

Medicinal Value ◽

Long Time ◽

Sequencing Platforms

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.

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

GigaScience ◽

10.1093/gigascience/giy009 ◽

2018 ◽

Vol 7 (3) ◽

Cited By ~ 26

Author(s):

Rachael E Workman ◽

Alexander M Myrka ◽

G William Wong ◽

Elizabeth Tseng ◽

Kenneth C Welch ◽

...

Keyword(s):

Single Molecule ◽

Full Length ◽

Full Length Transcript ◽

Insight Into

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SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis

10.1101/064840 ◽

2016 ◽

Author(s):

Afif Elghraoui ◽

Samuel J Modlin ◽

Faramarz Valafar

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Molecule ◽

Genetic Basis ◽

Reference Genome ◽

Reference Sequence ◽

Smrt Sequencing ◽

Virulence Attenuation ◽

Sequencing Platforms ◽

Genome Comparisons ◽

Reference Genomes

AbstractThe genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

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Single-Molecule Real-Time Transcript Sequencing of Turnips Unveiling the Complexity of the Turnip Transcriptome

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401434 ◽

2020 ◽

Vol 10 (10) ◽

pp. 3505-3514

Author(s):

Hongmei Zhuang ◽

Qiang Wang ◽

Hongwei Han ◽

Huifang Liu ◽

Hao Wang

Keyword(s):

Real Time ◽

Brassica Rapa ◽

Single Molecule ◽

Developmental Stages ◽

Full Length ◽

Sequencing Data ◽

Smrt Sequencing ◽

High Quality ◽

Transcript Structure ◽

Novel Transcripts

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.

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