Identification of MicroRNAs and Their Targets Involved in Paeonia rockii Petal Variegation Using High-throughput Sequencing

in Journal of the American Society for Horticultural Science

Tree peony (Paeonia sp.) is a popular traditional ornamental plant in China. Among the nine wild species, Paeonia rockii displays wide-ranging, deep purple variegation at the base of the petals, whereas Paeonia ostii exhibits purely white petals. Overall, the posttranscriptional regulation involved in tree peony flower opening and pigmentation remains unclear. To identify potential microRNAs (miRNAs) involved in flower variegation, six small RNA libraries of P. ostii and P. rockii petals at three different opening stages were constructed and sequenced. Using Illumina-based sequencing, 22 conserved miRNAs and 27 novel miRNAs were identified in P. rockii and P. ostii petals. Seventeen miRNAs were differentially expressed during flower development, and several putative target genes of these miRNAs belonged to transcription factor families, such as Myb domain (MYB), and basic helix-loop-helix (bHLH) transcription factors. Furthermore, an integrative analysis of the expression profiles of miRNAs and their corresponding target genes revealed that variegation formation might be regulated by miR159c, miR168, miR396a, and novel_miR_05, which target the MYB transcription factors, chalcone synthase (CHS), and ABC transporter. Our preliminary study is the first report of miRNAs involved in Paeonia flower pigmentation. It provides insight regarding the molecular mechanisms underlying the regulation of flower pigmentation in tree peony.

Contributor Notes

This work was supported by the Natural Science Foundation of China (31800599), Natural Science Foundation of Shannxi Province, China (2017JQ3024), General Financial Grant from the China Postdoctoral Science Foundation (2017M623267), the Special Fund for Scientific Research in the Public Welfare (201404701), the Scientific Startup Foundation for Doctors of Northwest A&F University (Z109021611), and Fundamental Research Funds for the Central Universities (Z109021606).

Qianqian Shi and Xiaoxiao Zhang performed the experiments and data analysis and wrote the manuscript. Xiang Li performed some experiments. Xiaoning Luo, Jianrang Luo, and Lixia He helped prepare the plant material. Yanlong Zhang and Long Li designed this research study. All of the authors read and approved the final manuscript.

These authors contributed equally to this work and should be considered co-first authors.

Corresponding authors. Email: zhangyanlong@nwsuaf.edu.cn or lilong1949@126.com.

Article Sections

Article Figures

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    Photographs of Paeonia ostii and Paeonia rockii during five different flower opening stages. (AE) P. ostii at S1–S5. (FJ) P. rockii at S1–S5 (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

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    A study of the overall expression patterns of all miRNAs in the six miRNA libraries. (A) General distribution of the log10-transformed expression values [transcripts per million (TPM)] of all miRNAs in the six libraries. (B) Correlations among the six miRNA libraries based on the Pearson correlation coefficient. The color scale represents the Pearson correlation coefficients among the different samples. The higher the Pearson correlation coefficient, the closer the relationship between the two libraries. Blue represents a close relationship; pink represents a distant relationship.

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    A study of Venn diagrams of all miRNAs between Paeonia ostii and Paeonia rockii (A) and differentially expressed miRNAs at different flower opening stages (B). S1, S3, and S5 represent three different developmental stages (stage 1, stage 3, and stage 5, respectively) in P. ostii and P. rockii (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

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    The study of expression patterns of the differentially expressed conserved and novel miRNAs among the three different opening stages of Paeonia rockii (PR) and Paeonia. ostii (PO) based on Illumina (San Diego, CA) sequencing datasets using Heml software (Deng et al., 2014). The bar indicates the log2-transformed expression level scale of the miRNAs. Each column represents a sample, and the colored bar indicates the relative expression level from high (red) to low (black). S1, S3, and S5 represent three different developmental stages (stage 1, stage 3, and stage 5, respectively) in PO and PR (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

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    The study of expression profiles and the fold changes of six miRNAs and their corresponding target genes in petals at five different flower opening stages Paeonia rockii (PR) (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers) in PR and Paeonia ostii (PO) using qRT-PCR. The expression profiles of six miRNAs and their corresponding target genes and the fold changes (PR/PO) of six miRNAs and their corresponding target genes are shown. U6 snRNA was used as a reference for the miRNA qRT-PCR. TUB was chosen as the internal housekeeping gene control for the target genes. Gene and miRNA expression levels at the S1 stage of PO were selected as the internal control, and the expression level of all genes in the control was set at 1.0. Error bars indicate the sd of three biological replicates and three technical replicates. (A) miR159c and c115438.graph_c0 (Myb DNA-binding protein, MYB1). (B) miR168 and c105628.graph_c0 (Myb DNA-binding protein, MYB2). (C) miR319 and c194399.graph_c0 (Myb DNA-binding protein, MYB3). (D) novel_miR_05 and c121983.graph_c0 (Myb DNA-binding protein, MYB4). (E) novel_miR_05 and c119993.graph_c0 (Myb DNA-binding protein, MYB5). (F) miR396a and c130130.graph_c0 (helix-loop-helix DNA-binding domain, bHLH1). (G) miR396b and c103359.graph_c0 (helix-loop-helix DNA-binding domain, bHLH2). (H) miR168 and c43510.graph_c0 (chalcone synthase, CHS). (I) miR396a and c129851.graph_c0 (ATP-binding glutathione S-conjugate). All miRNAs and their target genes are listed in Supplemental Table 1. The correlations (Cor) between miRNAs and targets were calculated based on fold changes of miRNA and the target gene expression between PR and PO using the Pearson correlation coefficient using SPSS (version 19.0; IBM Corp., Armonk, NY).

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    The study of the length distribution of miRNAs identified in Paeonia rockii and Paeonia ostii petals. The x-axis represents the length of miRNAs. The y-axis represents the number of miRNAs with a specific length.

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    Predicted precursor structures of all identified miRNAs. (A) Conserved miRNAs. (B) Novel miRNAs. Red sequences represent the mature sequences. Yellow sequences represent the loop structure. Purple sequences represent the star sequences predicted by miRDeep2 software. Blue sequences represent the star sequences from reads by sRNA sequencing.

  • View in gallery

    The study of correlations between the gene expression results obtained by qRT-PCR analysis and those obtained using Illumina sequencing for the 32 identified miRNAs at three different opening stages (S1, S3, and S5) in Paeonia ostii and P. rockii petals. The correlations were analyzed based on the average log2 values of expression levels of each miRNA during the three opening stages of P. ostii and P. rockii (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

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