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

doi:10.21203/rs.2.17990/v1

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

https://doi.org/10.21203/rs.2.17990/v1

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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’.

Epigenetics & Genomics

Chrysanthemum morifolium

transcriptome

SMRT-Seq

flavonoid

Dried inflorescence of Chrysanthemum morifolium Ramat. (Asteraceae) have been widely used in China for thousands of years and have significant medicinal and economic value. According to the Chinese Pharmacopoeia, medicinal C. morifolium can be mainly classified into five varieties, namely, ‘Hangju’, ‘Boju’, ‘Chuju’, ‘Gongju’ and ‘Huaiju’. Many pharmacological studies have reported that medicinal C. morifolium has antioxidative [1-3], cardiovascular protective [4, 5], antimicrobial [6] and anti-inflammatory [7, 8] activities, which are mainly due to its active components, such as flavonoids. The C. morifolium variety ‘Hangju’ has been cultivated for centuries and includes several genotypes, with the genotype ‘Zaoyang’ having the largest cultivated area and yield [9]. Furthermore, the genotype ‘Zaoyang’ from Tongxiang County (the traditional cultivation area) is considered superior to genotypes from other cultivation areas due to its high luteolin content [10], which suggests that flavonoids are the vital components in genuine medicinal C. morifolium.

Flavonoids are a large group of secondary metabolites in plants whose biosynthetic pathways are well established as useful models for studying metabolic regulation. The genes involved in the flavonoid biosynthesis pathways are mainly divided into early and late biosynthetic genes according to their location in the pathways. Early biosynthetic genes (EBGs) including CHS, CHI, F3H, F3’H and FLS catalyse flavonol biosynthesis, whereas late biosynthetic genes (LBGs: DFR, ANS and ANR) lead to both anthocyanin and proanthocyanidin biosynthesis [11-13]. Other genes such as FNS and IFS can also catalyse flavone and isoflavone biosynthesis [14, 15]. Furthermore, the transcription factors regulating such biosynthesis have been identified in many species. AtMYB11, AtMYB12 and AtMYB111 in Arabidopsis thaliana activate the expression of LEGs to produce flavonol [16, 17], and high expression of MdMYB1 and MdMYB10 increases the anthocyanin content in Malus × domestica [18]. However, flavonoid biosynthesis and regulation in C. morifolium are poorly understood because of the lack of a reference genome database. Thus, transcriptome sequencing offers an alternative approach for obtaining gene information. Second-generation sequencing (SGS) was used to identify the differentially expressed genes in C. morifolium under stress or in different tissues [19-22]. However, the short-length reads generated by SGS decrease the accuracy of transcript assembly, especially for species without genome information. Single-molecule real-time (SMRT) sequencing, as a third-generation sequencing (TGS) technology, has the advantage of generating high-throughput long reads and thus avoids the inaccuracy resulting from assembling short sequences [23, 24].

In this study, SMRT sequencing was performed to obtain full-length transcripts for better understanding flavonoid biosynthesis and regulation in C. morifolium. The key genes were screened based on annotation analysis, and their expression profiles were analysed at different developmental stages. Furthermore, the candidate MYB transcription factors activating flavonol biosynthesis were identified, which not only provide an understanding of flavonoid biosynthesis and regulation in C. morifolium but also will advance the introduction and cultivation of C. morifolium.

The full-length transcriptome of C. morifolium

To identify the transcripts in C. morifolium during the developmental stages, SMRT sequencing technology was used to obtain the complete and full-length transcriptomes. In total, we obtained 15.56 Gb and 13.93 Gb of raw data from the 0-4.5K and 4.5-10K libraries, respectively. Approximately 591,513 and 560,454 ROIs (reads of insert) were recognized, with mean lengths of 3,328 and 3,587 bp, respectively (Table 1). The clean data were uploaded to the Sequence Read Archive (SRA) with accession numbers SRR10054190 and SRR10054977. More than 50% of the reads were classified as full-length (FL) non-chimeric reads, and fewer than 5% of the reads were classified as chimeric reads, which indicated that the SMRTbell libraries were of high quality (Supplementary figure 1). After clustering and polishing, 129,610 and 112,142 HQ isoforms were finally merged into 125,532 non-redundant isoforms with a mean length of 2,009 bp and an N50 of 2,274 bp (Table 1).

Functional annotation

All non-redundant isoforms were searched against 7 functional databases, and approximately 115,807 (92.25%) transcripts were annotated in at least one functional database (Figure 2A). There were 107,700 (85.79%) isoforms assigned to NR databases, and most of them had high similarity to genes in Helianthus annuus (60.13%), Cynara cardunculus var. scolymus (24.18%), Chrysanthemum × morifolium (1.66%) and other species (Figure 2B). Approximately 33,901 (27.01%) transcripts were assigned to 53 subcategories in the GO database, and the largest subcategories of the three primary GO categories were cellular process, cell and catalytic activity (Supplementary figure 2). We also obtained annotations for 91,179 (72.63%) isoforms from the KEGG database, 2,411 of which were associated with the Biosynthesis of other secondary metabolites subgroup. Furthermore, we found that 1,447, 480, 54 and 102 transcripts were annotated as belonging to the Phenylpropanoid biosynthesis, Flavonoid biosynthesis, Anthocyanin biosynthesis, Isoflavonoid biosynthesis, and Flavone and flavonol biosynthesis pathways, respectively (Figure 2C).

The candidate genes involved in the flavonoid biosynthesis pathway

Combining the annotation results from seven databases, 143 isoforms were annotated as PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, FLS, FNS, DFR and ANS genes in the flavonoid biosynthesis pathways (Table 2). Every gene had many isoforms, especially those in the PAL and 4CL gene families, with more than 30 isoforms. Interestingly, no sequences were similar to F3’5’H, which catalyses the substrate of blue anthocyanins. This finding is consistent with the lack of blue varieties of medicinal C. morifolium. ANR and LAR genes also were not found in the database, which suggested a lack of proanthocyanins in medicinal C. morifolium. Further analysis of the genes involved in luteolin and quercetin biosynthesis revealed that the isoforms belonged to 5 CHS genes, 3 CHI genes, 1 F3H gene, 1 F3’H gene, 1 FNS Ⅱ gene and 2 FLS genes based on sequence similarity (Supplementary table 1).

Differential expression of genes involved in luteolin and quercetin biosynthesis

Luteolin and quercetin are the marker compounds of ‘Hangju’ in the Chinese Pharmacopoeia; thus, the CHS, CHI, F3H, F3’H, FLS and FNS genes were selected and analysed. The expression profile of every gene at the different flowering stages is shown in Figure 3. Most of the genes had the highest expression level at the bud stage (stage A), which is an important stage for flavonoid biosynthesis. Expression of CHS1 was significantly higher than that of the other CHS genes at every stage, while CHS4 and CHS5 were barely expressed. Therefore, CHS1 might be a major gene controlling flavonoid production in the inflorescence of C. morifolium. Similarly, CHI1, CHI3 and FLS1 may also be key genes. Interestingly, the CHS2 gene had a high expression level at stage A but was not expressed at the other stages. The change in expression level suggested that CHS2 has significant stage-specific expression.

The MYBs regulating flavonoid biosynthesis in C. morifolium

Based on their protein domains, a total of 4,068 isoforms were identified as transcription factors, and MYB was the largest family, with approximately 444 isoforms (Figure 4A). After alignment with MYB transcription factors isolated from A. thaliana, we found that 17 isoforms were clustered into R2R3-MYB subgroups 4-7 (Supplementary figure 3). The members of all four subgroups also aligned with other MYBs isolated from Malus × domestica, Vitis vinifera and other species (Figure 4B); the information on the genes is provided in Supplementary table 2. Finally, three isoforms were clustered into R2R3 MYB subgroup 7, members of which are regarded as activators of flavanol biosynthesis. Five isoforms were grouped into subgroup 6, members of which have an active function in anthocyanin biosynthesis. Nine isoforms were grouped into subgroup 4, members of which repress the biosynthesis of phenylpropanoids, such as lignin, phenolic acid and flavonoids. However, no isoforms were clustered with subgroup 5, members of which activate the biosynthesis of proanthocyanidins. This is consistent with the result that no isoforms were annotated as LAR and ANR genes. Furthermore, we found that isoforms 90494 and 90874 had high similarity to a single gene, which was not similar to isoform 65995; therefore, we inferred that two genes activated flavanol biosynthesis. In addition, the isoforms in subgroups 4 and 6 were assigned to 2 genes and 1 gene, respectively, based on sequence similarity (Supplementary table 3). When comparing the expression levels of these two genes in the inflorescence, we found that CmMYBF1 was more highly expressed than CmMYBF2 at every stage (Figure 4C) and CmMYBF1 had an expression profile similar to those of FLS genes. Therefore, CmMYBF1 probably activates flavonoid biosynthesis in the inflorescence of C. morifolium.

C. morifolium, as a traditional medicinal plant, has a long culture history in China, and its inflorescence has high medicinal value due to its flavonoids. However, there is a poor understanding of flavonoid biosynthesis and regulation in C. morifolium. To identify genes involved in flavonoid biosynthesis and regulation with long sequences, full-length transcriptome sequencing of the inflorescence during flowering was performed. By comparison with 63,854 unigenes with a mean length of 741 bp isolated from ‘Chuju’ by SGS technology, we found 125,532 isoforms in ‘Hangju’ with a significantly longer mean length of 2,009 bp. We found more genes in the flavonoid biosynthesis pathways in this cultivar than reported in ‘Chuju’ [25]. SMRT sequencing was clearly more accurate than SGS technology in the process of obtaining transcript information. Furthermore, TGS is also an effective method for detecting alternative splicing (AS) events [26, 27]. In this study, we found that many isoforms shared high similarity with some fragment inserts or losses. Therefore, these isoforms were perhaps the results of AS of one gene.

According to the gene annotations, we found 5 CHS and 3 CHI genes in the database for C. morifolium, fewer than the 17 CHS and 8 CHI genes identified in the genome of Chrysanthemum nankingense [28]. Moreover, no LAR or ANR genes also were detected in Chrysanthemum nankingense, which suggests that proanthocyanidins are not the functional component in C. morifolium and C. nankingense. In addition, C. morifolium has a gene number similar to that in other species of Asteraceae. For instance, sunflower also has 3 CHI genes, 2 FLS genes and 1 F3H gene, similar to C. morifolium [28]. In our previous study, one FLS gene was reported in C. morifolium to catalyse dihydroquercetin to quercetin [29]. In addition, we found that another FLS gene was highly similar to the predicted gene in Lactuca sativa and Nicotiana tabacum, which needs to be verified in future studies. At the different stages, the genes had a high expression level at the bud stage, CHS, CHI, F3H, F3’H and FLS genes also highly expressed in budding of ‘Chuju’ [25].

In the MYB transcription factor analysis, the 5 transcripts in subgroup 6 were annotated as the CmMYB6 gene (No.: AKP06190), which reportedly functions as the activator of anthocyanin biosynthesis in C. morifolium [30]. The ternary MYB-bHLH-WD40 (MBW) transcriptional regulator was recognized to regulate flavonoid biosynthesis, especially anthocyanin and proanthocyanidin biosynthesis [31, 32]. However, no factors in C. morifolium were clustered with bHLH (AtEGL3, AtGL3 and TT8), which interacts with MYB to regulate anthocyanin biosynthesis (Supplementary figure 4) [33, 34]. In addition, no genes were annotated as the CmbHLH2 gene (No.: KT724056), which reportedly functions in the activation of anthocyanin biosynthesis in C. morifolium for ornamentation [35]. TT8 also promotes proanthocyanidin biosynthesis [31], and no genes were similar to TT8 result in the small amounts of anthocyanins and proanthocyanidins produced in ‘Hangju’. Therefore, it reliable to find candidate transcription factors via phylogenetic analysis. Accordingly, we found two genes belonging to SG7 MYBs, which are important factors activating flavonol biosynthesis. Both the CmMYBF1 and CmMYBF2 proteins had a GR[V/T]SR[C/S][V/K][A/M]K motif, which is the typical motif of SG7 MYB transcription factors [36]. Furthermore, CmMYBF2 gene had a sequence similar to that of the CmMYB3 gene, which has been proposed to regulate flavonoid biosynthesis but not promote DRF gene expression in C. morifolium for ornamentation [30]; thus, CmMYBF2 very likely regulates flavonol biosynthesis. However, CmMYBF1 had a higher expression level than CmMYBF2, and CmMYBF1 might also be a transcription factor regulating flavonol biosynthesis. Thus, which transcription factor is the most important needs to be further verified.

Conclusion

In our study, the TGS platform (PacBio Sequel) was used to obtain the full-length transcriptome of C. morifolium cv. ‘Hangju’. A total of 29.49 Gb of raw data and 125,532 non-redundant isoforms were obtained. After gene annotation, 144 isoforms involved in the pathway of flavonoid biosynthesis were detected. We also predicted the major genes participating in luteolin and quercetin biosynthesis by expression analysis. The candidate genes had similar expression patterns, with the highest expression level at the bud stage. Furthermore, the MYB transcription factors activating flavonol biosynthesis were screened and analysed. All of these results provide a theoretical basis for further studying the quality of ‘Hangju’ and will advance its introduction and cultivation.

Plant material and RNA extraction

The ‘Zaoyang’ genotype of Chrysanthemum morifolium cv. ‘Hangju’ was formally identified by Professor Qiaosheng Guo and collected from the germplasm resource preservation centre at Nanjing Agricultural University. The plant material used in our study is recognized by the Department of Agriculture of Zhejiang Province, China, but no voucher specimens have been deposited in public herbaria. The inflorescence at different developmental stages (stage A – stage E) were collected and frozen at -80°C for subsequent experiments (Figure 1) [10, 37]. Total RNA was isolated from the samples by using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, China). The quality of RNA was assessed on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose gel. Then, equal amounts of high-quality RNA from every sample were pooled together for PacBio sequencing analysis.

PacBio SMRTbell library preparation and sequencing

A total of 800-1,000 ng of RNA was used to synthesize cDNA by a Clontech SMARTer PCR cDNA Synthesis Kit with oligo-dT primers. After large-scale PCR amplification, the double-stranded cDNA was size selected into 0-5 kb and 4.5-10 kb fractions by the BluePippin system (Sage Science, USA). Additional PCR was performed post size selection to produce adequate templates, and then, size-selected cDNA was ligated with a blunt adapter for SMRTbell library construction. Finally, a total of two SMRT cell sequencing reactions were performed by a PacBio Sequel sequencer (BGI-Shenzhen, China).

Bioinformatics analysis of isoform sequences

Reads of insert were generated by removing adapters and artefacts from raw SMRT sequencing data, and they were further classified into FL non-chimeric, FL chimeric, non-FL, or short reads based on the presence of 5’/3’ primer and polyA-tail sequences. Then, FL non-chimeric transcripts were clustered into consensus sequences by the ICE (Interactive Clustering and Error Correction) algorithm and polished by Quiver. Finally, high-quality consensus isoforms from each library were merged, and CD-HIT-EST was used to remove redundant isoforms based on sequence similarity. The isoform sequences were aligned to the non-redundant protein (NR), non-redundant nucleotide (NT), Gene Ontology (GO), Clusters of Orthologous Groups (COG), Kyoto Encyclopaedia of Genes and Genomes (KEGG), SwissProt, and InterPro databases with Blast, Blast2GO and InterProScan 5. TransDecoder was used to identify the candidate coding areas and extract the longest ORF. Then, the homologous protein sequences were searched against SwissProt and Hmmscan to predict the coding regions.

Expression analysis of flavonoid biosynthesis genes in C. morifolium

According to functional annotation analysis, genes involved in the luteolin and quercetin biosynthesis pathways were selected (including CHS, CHI, F3H, F3’H, FLS, and FNS). Then, the predicted CDSs were used to design primers (Supplementary table 4), and their expression profiles at different developmental stages were analysed by Q-PCR with relative quantification.

Phylogenetic analysis of the MYB transcription factor family

The ORFs of isoforms were searched against the PlantTFDB databases by hmmsearch to identify MYB transcription factors. Then, the protein sequences of putative MYBs were aligned with 168 MYB transcription factors isolated from A. thaliana by MAFFT. The output file from MAFFT was used to construct a phylogenetic tree by RAxML with the maximum likelihood method and 1000 bootstraps. Then, candidate transcripts were further aligned with the MYB transcription factors regulating flavonoid biosynthesis in A. thaliana and other plants. The expression levels of candidate MYB genes were also analysed by Q-PCR.

List of abbreviations

SMRT-Seq: Single-molecule real-time sequencing; EBGs: Early biosynthetic genes; LBGs: Late biosynthetic genes; SGS: Second-generation sequencing; TGS: Third-generation sequencing; ROI: Reads of insert; NR: non-redundant protein; NT: non-redundant nucleotide; GO: Gene Ontology; COG: Clusters of Orthologous Groups; KEGG: Kyoto Encyclopaedia of Genes and Genomes; ICE: Interative Clustering and Error Correction; PAL: Phenylalanine ammonia-lyase; C4H: Cinnamate 4-hydroxylase; 4CL: 4-coumarate--CoA ligase; CHS: Chalcone synthase; CHI: Chalcone isomerase; F3H: Flavanone 3-hydroxylase; F3’H: Flavonoid 3’-hydroxylase; F3’5’H: Flavonoid 3’,5’-hydroxylase; FNS Ⅱ: Flavone synthase Ⅱ; IFS: Isoflavone synthase; FLS: Flavonol synthase; DFR: Dihydroflavonol 4-reductase; ANS: Anthocyanidin synthase; ANR: Anthocyanidin reductase; LAR: Leucoanthocyanidin reductase; MBW: MYB-bHLH-WD40; SG: Subgroup. ORF: Open read frame; AS: Alternative splicing. FL: Full length.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to the data was analyzing for other research, but are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by grants from the National Nature Science Foundation of China (No. 81503180) in the design of the study and collection of data. The Fundamental Research Funds for the Central Universities (No. KYZ201608 and KJQN201643) were used in supporting the analysis and interpretation of data and manuscript writing.

Authors' contributions

TW, FY, and QG conceived and designed the experiments. TW and FY performed the experiments and written the original draft. TW, FY, and QZ analyzed the data and make figures and tables. WZ and LZ contributed to cultivation and collection of experimental materials. All authors read and approved the final manuscript.

Acknowledgements

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Table 1 Statistics of transcriptome data
Library	B01 (0-5K)	G01 (4.5-10K)
Cell Number	1	1
Total Raw Reads	606,174	575,319
Total Base (GB)	15.56	13.93
Reads of Insert	591,513	560,454
Read Bases of Insert (bp)	1,823,985,219	2,010,084,287
Mean Read Length of Insert (bp)	3,084	3,587
HQ Isoforms Number	129,610	112,142
Total Iosforms Number	125,532
Mean Read Length of Isoforms (bp)	2,009
N50 (bp)	2,274

Table 2 The Isoforms annotated into the flavonoid biosynthesis pathways
Gene name	Numuber of Isoforms	Isoforms name
PAL	45	Isoform_52833, Isoform_47763, Isoform_43454, Isoform_62542, Isoform_67724, Isoform_102200, Isoform_108441, Isoform_81892, Isoform_68061, Isoform_25536, Isoform_27366, Isoform_28938, Isoform_35394, Isoform_27213, Isoform_26269, Isoform_110994, Isoform_26254, Isoform_122495, Isoform_121987, Isoform_121852, Isoform_57291, Isoform_48658, Isoform_45373, Isoform_55744, Isoform_76695, Isoform_79590, Isoform_94611, Isoform_88194, Isoform_68496, Isoform_90394, Isoform_95170, Isoform_77862, Isoform_102606, Isoform_22996, Isoform_33822, Isoform_29134, Isoform_38700, Isoform_39651, Isoform_112668, Isoform_102808, Isoform_28794, Isoform_123285, Isoform_43973, Isoform_50840, Isoform_50978
C4H	9	Isoform_61275, Isoform_63752, Isoform_89464, Isoform_51120, Isoform_51919, Isoform_73975, Isoform_62853, Isoform_53493, Isoform_55308
4CL	33	Isoform_43749, Isoform_59715, Isoform_42120, Isoform_44780, Isoform_79349, Isoform_89705, Isoform_110940, Isoform_70800, Isoform_80544, Isoform_74817, Isoform_33855, Isoform_104805, Isoform_40088, Isoform_46710, Isoform_40758, Isoform_45456, Isoform_48895, Isoform_64143, Isoform_42313, Isoform_49751, Isoform_52298, Isoform_91185, Isoform_73851, Isoform_75666, Isoform_113605, Isoform_19191, Isoform_48574, Isoform_39980, Isoform_40022, Isoform_123654, Isoform_57545, Isoform_46926, Isoform_50728
CHS	16	Isoform_69087, Isoform_88884, Isoform_70059, Isoform_71639, Isoform_67309, Isoform_90473, Isoform_71339, Isoform_70758, Isoform_72050, Isoform_79285, Isoform_69249, Isoform_57251, Isoform_58137, Isoform_71025, Isoform_63828, Isoform_84387
CHI	8	Isoform_101012, Isoform_107006, Isoform_107139, Isoform_108103, Isoform_108228, Isoform_111718, Isoform_91714, Isoform_92657
F3H	3	Isoform_82489, Isoform_122564, Isoform_84881
F3'H	6	Isoform_50027, Isoform_74567, Isoform_86056, Isoform_54616, Isoform_108195, Isoform_124366
FLS	2	Isoform_82400, Isoform_83438
FNS II	13	Isoform_52845, Isoform_102292, Isoform_74378, Isoform_84302, Isoform_54094, Isoform_49130, Isoform_75446, Isoform_83978, Isoform_112139, Isoform_52828, Isoform_94894, Isoform_101125, Isoform_110531
DFR	5	Isoform_34792, Isoform_82000, Isoform_76056, Isoform_77859, Isoform_119850
ANS	3	Isoform_86095, Isoform_83405, Isoform_100339

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

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