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Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling

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Abstract

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Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.).

Abstract

Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

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Acknowledgments

S. Asha gratefully acknowledges the Senior Research Fellowship from Council of Scientific and Industrial Research (CSIR) New Delhi, Government of India. This work was financially supported by Department of Biotechnology, Government of India, New Delhi.

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Correspondence to E. V. Soniya.

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This article does not contain any studies with human participants performed by any of the authors.

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Additional information

Communicated by Z. Zhang.

Electronic supplementary material

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Supplementary material 1 Fig. S1 Flow chart of the methodology adopted to identify P. nigrum miRNAs (TIFF 96 kb)

299_2015_1866_MOESM2_ESM.pdf

Supplementary material 2 Fig. S2 Precursor structures of conserved miRNAs predicted from black pepper transcriptome Hair-pin secondary structure is highlighted with mature miRNA (red colour) and star sequence (blue colour). The MFE values of the predicted precursors were also mentioned. (PDF 199 kb)

299_2015_1866_MOESM3_ESM.pdf

Supplementary material 3 Fig. S3 miRNA isoform members of known miRNA families, their reads and alignments to corresponding precursors MiRNA candidates located in the 5p and 3p are numbered. Mature miRNA and miRNA* are highlighted with red and blue colours (PDF 302 kb)

Supplementary material 4 Fig. S4 Reverse complimentary miRNAs identified from black pepper (TIFF 181 kb)

Supplementary material 5 (XLS 21 kb)

Supplementary material 6 (DOC 262 kb)

Supplementary material 7 (DOC 104 kb)

Supplementary material 8 (XLS 67 kb)

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Asha, S., Sreekumar, S. & Soniya, E.V. Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling. Plant Cell Rep 35, 53–63 (2016). https://doi.org/10.1007/s00299-015-1866-x

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  • DOI: https://doi.org/10.1007/s00299-015-1866-x

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