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Profiling of microRNAs in wild type and early flowering transgenic Chrysanthemum morifolium by deep sequencing

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Abstract

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.

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Abbreviations

AMFE:

Adjusted MFE

AP:

APETALA

ARF:

AUXIN RESPONSE FACTOR

CO:

CONSTANS

Ct:

Cycle threshold

DREB:

Dehydration responsive element binding protein

FLC:

FLOWERING LOCUS C

FT:

FLOWERING LOCUS T

FUL:

FRUITFULL

GA:

Gibberellin

GO:

Gene ontology

LFY:

LEAFY

MFE:

Minimal folding free energy

MFEI:

MFE index

NGS:

Next-generation sequencing

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

SEP:

SEPALLATA

SOC1:

SUPRESSOR OF OVEREXPRESSION OF CO1

SPL:

SQUAMOSA-PROMOTER BINDING-LIKE-LIKE

SVP:

SHORT VEGETATIVE PHASE

TFL1:

TERMINAL FLOWER1

WT:

Wild type

SD:

Short day

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Acknowledgements

This work was supported by the grant from the Russian Foundation for Basic Research 12-07-31039-MOL_A_2012, President Grant for Government Support of Young Russian Scientist–2012 (SP-2056.2012.5), the Program of the Presidium of the Russian Academy of Sciences “Molecular and Cell Biology” (01201353319), and was performed using the experimental climate control facility.

Author contributions

EBP and KGS conceived and designed the experiments. AVN and ESP performed the experiments. Analyzed the data: OAS, AVS, AVN, FSS, AAS, SMR, ASS, and NMG analyzed the data. EBP and AAS contributed reagents, materials, and analytic tools. OAS and AVS provided plant material. OAS, AVS, and AVN wrote the paper. All authors read and approved the final manuscript.

Author information

Correspondence to O. A. Shulga.

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Conflict of interest

The authors declare that they have no conflict of interest.

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Supplementary material 1 ESM_1 Supplementary Tables S1–S14 ‘Summary of high-throughput sequencing of C. morifolium small RNA library. S1 – sRNA library WT_leaf (S1), sequencing dataset, 100% homology (0 mismatches, mm); S2 – sRNA library WT_ap (S5), sequencing dataset, 100% homology; S3 – sRNA library WT_infl (S9), sequencing dataset, 100% homology; S4 – sRNA library 92-31_leaf (S2), sequencing dataset, 100% homology; S5 – sRNA library 92-31_ap (S6), sequencing dataset, 100% homology; S6 – sRNA library 92-31_infl (S10), sequencing dataset, 100% homology. S7 – sRNA library WT_leaf (S1), sequencing dataset, 0–2 mm; S8 – sRNA library WT_ap (S5), sequencing dataset, 0–2 mm; S9 – sRNA library WT_infl (S9), sequencing dataset, 0–2 mm; S10 – summary of normalized counts of sRNA libraries S1–S10, 0 mm; S11 – results of phylogenetic analysis of identified cmo-miRNAs; S12 – list of iso-cmo-miRNAs with different length compared to cmo-miRNAs; S13 – list of iso-cmo-miRNAs with different length and substitutions compared to cmo-miRNAs; S14 – list of putative novel chrysanthemum-specific miRNAs; S15 – results of comparison of previously reported chrysanthemum miRNAs with identified cmo-miRNAs; S16 – Results and bioinformatic validation of RT-qPCR (Ct) obtained for 10 cmo-miRNAs in 12 WT and transgenic chrysanthemum samples; S17 – candidate target mRNAs for identified mature conserved cmo-miRNAs; Gene Ontology categorization; S18 – candidate target mRNAs for identified putative novel cmo-miRNAs; Gene Ontology categorization; S19 – Primers used for stem-loop RT-qPCR analysis of miRNA expression, and expression stability of candidate reference cmo-miRNAs across different chrysanthemum tissues’ (XLSX 1446 KB).

Supplementary material 2 ESM_2 Supplementary Table S20 ‘Known conserved mature miRNA and miRNA* in C. morifolium’ (PDF). (PDF 1587 KB)

Supplementary material 3 ESM_3 Supplementary Figures S1–S9 ‘cmo-miRNAs differential expression and alignment. S1 – Prediction of expression stability for 10 C. morifolium miRNAs by NormFinder. The least stable miRNAs are on the left (green), and the most stable miRNAs are on the right (red); S2 – expression of 10 cmo-miRNAs in leaves by RT-qPCR; S3 – expression of 10 cmo-miRNAs in the apex by RT-qPCR; S4 – expression of 10 cmo-miRNAs in the inflorescence by RT-qPCR; S5 – expression of 10 cmo-miRNAs in the leaf, apex, and inflorescence of the WT plant by RT-qPCR; S6 – expression of 9 selected cmo-miRNAs in the inflorescence by NGS counts (in sequenced libraries) normalized om cmo-miR162a value; S7 – expression of selected cmo-miRNAs by counts (in sequenced libraries); S8 – Sequence alignment of cmo-miR6111.2 and cca-miR6111 precursors; cmo-miR6111/6111* and cmo-miR6111.2/6111.2* positions are indicated; cca-miR6111-5p and 3p are highlighted in red; S9 – Comparison of expression dynamics of cmo-miRNAs during flowering progression from initiation to anthesis in WT and transgenic chrysanthemums. (a) Comparison of NGS evaluated miRNA expression in WT and 92-31 plants. (b) Comparison of RT-qPCR evaluated miRNA expression in ordinarily flowering WT and 111-1 plants. (c) Comparison of RT-qPCR evaluated miRNA expression in early flowering 92-31 and 111-2 plants. The central part includes cmo-miRNAs with similar expression dynamics in both compared plants. The left and right parts represent cmo-miRNAs with certain plant-specific dynamics. The cmo-miRNAs upregulated, downregulated, or maintaining constant expression levels during flowering progression, are shown at the top, bottom, or middle. cmo-miRNAs are given in corresponding numbers’ (XLS). (XLSX 1997 KB)

Supplementary material 4 ESM_4 Supplementary Table S21 ‘Characteristics of precursors for the candidate novel and conserved miRNAs identified in C. morifolium’ (PDF 1417 KB)

Supplementary material 5 ESM_5 Supplementary Figure S10 ‘Secondary structures of known conservative (10-1 – 10-4) and candidate novel (10-5 – 10-34) pre-cmo-miRNAs. 10-1 shows pre-cmo-miR156p; 10-2 – pre-cmo-miR477g; 10-3 – pre-cmo-miR6117; 10-4a – pre-cmo-miR6111.2; 10-4b – pre-cca-miR6111; 10-5 – pre-cmo-miR005; 10-6 – pre-cmo-miR007; 10-7 – pre-cmo-miR008; 10-8 – pre-cmo-miR009; 10-9 – pre-cmo-miR011; 10-10 – pre-cmo-miR013; 10-11 – pre-cmo-miR014; 10-12 – pre-cmo-miR015; 10-13 – pre-cmo-miR018; 10-14 – pre-cmo-miR020; 10-15 – pre-cmo-miR024; 10-16 – pre-cmo-miR025; 10-17 – pre-cmo-miR026; 10-18 – pre-cmo-miR027; 10-19 – pre-cmo-miR028; 10-20 – pre-cmo-miR031; 10-21 – pre-cmo-miR032; 10-22 – pre-cmo-miR033; 10-23 – pre-cmo-miR039; 10-24 – pre-cmo-miR045; 10-25 – pre-cmo-miR046; 10-26 – pre-cmo-miR047; 10-27 – pre-cmo-miR048; 10-28 – pre-cmo-miR052; 10-29 – pre-cmo-miR053; 10-30 – pre-cmo-miR060; 10-31 – pre-cmo-miR062; 10-32 – pre-cmo-miR065; 10-33 – pre-cmo-miR075; 10-34 – pre-cmo-miR077’ (PDF) (PDF 2560 KB)

Supplementary material 6 ESM_6 Supplementary Table S22 ‘In silico predicted mRNA targets for Asteraceae-specific, conserved and novel cmo-miRNAs’ (PDF) (PDF 1110 KB)

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Shulga, O.A., Nedoluzhko, A.V., Shchennikova, A.V. et al. Profiling of microRNAs in wild type and early flowering transgenic Chrysanthemum morifolium by deep sequencing. Plant Cell Tiss Organ Cult 128, 283–301 (2017). https://doi.org/10.1007/s11240-016-1109-z

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Keywords

  • MicroRNA
  • Chrysanthemum morifolium
  • Deep sequencing
  • Flowering time