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Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencing

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Abstract

Anthocyanins are widely distributed water-soluble phytochemical pigments belonging to the flavonoid group. To date, limited knowledge is available about the regulatory roles of miRNAs in anthocyanin biosynthesis in plants. To identify the miRNAs associated with anthocyanin biosynthesis in radish, five small RNA (sRNA) libraries constructed from ‘Xinlimei’ radish roots at 11, 21, 44, 56 and 73 days (d) were examined using high-throughput sequencing technology. A total of 102.02 million (M) clean reads were generated, from which 483 known and 1415 novel miRNAs were identified. Combined with target prediction and annotation, 72 differentially expressed miRNAs (52 known and 20 novel miRNAs) were more likely to participate in anthocyanin biosynthesis. Several target genes for these miRNAs encode a few transcription factors, including Myb domain (MYB), basic helix-loop-helix (bHLH), WD40 repeat, squamosa promoter binding protein like (SPL), auxin response factor (ARF), ethylene insensitive 3 (EIN3), WRKY and MADS-box proteins. Furthermore, the expression patterns of some anthocyanin biosynthesis related miRNAs and their corresponding targets were validated by RT-qPCR. Based on the characterization of anthocyanin biosynthesis related miRNAs and their target genes, a putative miRNA-target module regulating anthocyanin biosynthesis was proposed. This study represents the first genome-wide identification of miRNAs associated with anthocyanin biosynthesis in radish, and provides insights into the molecular mechanisms underlying regulation of anthocyanin biosynthesis in radish and other crops.

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Acknowledgements

This work was funded by the Ministry of Science and Technology of the People’s Republic of China (2013BAD01B04, 2012BAD02B01, and 2012AA021801-4), the Outstanding Talents Program and Innovation Program (Vegetable Germplasm Resources Team, CAAS-ASTIP-2013-IVFCAAS) of the Chinese Academy of Agricultural Sciences and the Key Laboratory of Horticultural Crops Genetic Improvement, Ministry of Agriculture of the People’s Republic of China.

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Authors and Affiliations

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Yuyan Sun, Yang Qiu, Mengmeng Duan, Jinglei Wang, Xiaohui Zhang, Haiping Wang, Jiangping Song & Xixiang Li

  2. Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China

    Yuyan Sun

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  1. Yuyan Sun
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  2. Yang Qiu
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  3. Mengmeng Duan
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Correspondence to Xixiang Li.

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Communicated by S. Hohmann.

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438_2016_1268_MOESM1_ESM.doc

Supplementary Table S1. Primers used for quantification and validation of selected miRNAs and their potential targets (DOC 40 kb)

Supplementary Table S2. Known miRNAs identified from five radish root libraries (XLS 86 kb)

Supplementary Table S3. Novel miRNAs precursor candidates identified for five radish root libraries (XLS 1360 kb)

Supplementary Table S4. Unique novel miRNA sequences generated from five radish root libraries (XLS 409 kb)

Supplementary Table S5. Differentially expressed known miRNAs among five radish root libraries (XLS 93 kb)

Supplementary Table S6. Differentially expressed novel miRNAs among five radish root libraries (XLS 64 kb)

Supplementary Table S7. Target prediction for differentially expressed miRNAs (XLS 213 kb)

438_2016_1268_MOESM8_ESM.tif

Supplementary Fig S1. Length distribution of small RNAs (sRNAs) in the libraries of the radish roots. The y-axis indicates the percentage of sRNA reads in each library, whereas the x-axis corresponds to the nucleotide (nt) length of sRNAs (TIFF 732 kb)

Supplementary Fig S2. Venn diagram of known miRNAs in the five libraries of radish roots (TIFF 1139 kb)

Supplementary Fig S3. Secondary structure prediction of novel miRNAs precursors (PDF 2047 kb)

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Sun, Y., Qiu, Y., Duan, M. et al. Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencing. Mol Genet Genomics 292, 215–229 (2017). https://doi.org/10.1007/s00438-016-1268-y

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