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Identification and characterization of microRNA expression in Ginkgo biloba L. leaves

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Abstract

Ginkgo biloba L., an extant primitive gymnosperm species, occupies an important position in the evolution of the plant kingdom. Furthermore, its leaves contain a large number of active, medicinally valuable compounds; therefore, it is also an important tree economically. MicroRNAs (miRNAs) are important regulators of gene expression implicated in controlling plant development and environmental interaction. Extensive studies of miRNAs have been performed in angiosperms, but no reports have focused on small RNAs in the basal gymnosperm G. biloba. Here, we used Illumina next-generation sequencing HiSeq™ 2000 platform to identify and quantitatively profile small RNAs in G. biloba leaves. A total of 13,414,548 and 12,293,511 reads, containing 202 and 201 known miRNAs, belonging to 82 and 78 families, were sequenced from two small RNA libraries constructed from female and male leaves, respectively. We also identified 174/174 novel miRNAs in the female/male leaves of G. biloba. Relative expression levels of the miRNAs and the precursor sequences of several of the known and novel miRNAs were further validated by subcloning and quantitative real-time PCR (qRT-PCR). Target predictions of the 380 and 340 known miRNAs, from female and male leaves, resulted in 2116 and 2002 target genes, while those of the 155 and 158 novel miRNAs resulted in 596 and 793 target genes, respectively. KEGG annotation analysis revealed that high-ranked miRNA-target genes are implicated in plant-pathogen interactions, plant hormone signal transduction, and flavonoid biosynthesis. Taken together, we identified a large number of miRNAs in mature female and male G. biloba leaves for the first time. We ascertained that the regulatory networks of the miRNAs are involved in many different primary biological processes based on potential target designations.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 31200145) and the Three New Forestry Engineering Foundation of Jiangsu Province (No. lysx[2013]05), (No. lysx[2014]12).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LW and BJ participated in the design of the study, performed the experimental work, and drafted the manuscript. JGZ and MZ contributed the bioinformatic analyses and drafted the manuscript, KGL, WXL, ZGL, and CQZ contributed the bioinformatic analyses. All authors read and approved the final manuscript.

Data archive statement

All of the raw and processed data of small RNA sequences in this research have been deposited in Gene Expression Omnibus (GEO) under the accession GSE65229.

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Authors and Affiliations

  1. College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, 225009, China

    Li Wang, Jianguo Zhao, Min Zhang, Weixin Li, Kaige Luo, Zhaogeng Lu & Biao Jin

  2. Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou, 225009, China

    Changquan Zhang

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  1. Li Wang
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  2. Jianguo Zhao
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  3. Min Zhang
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Corresponding author

Correspondence to Biao Jin.

Additional information

Communicated by A. Brunner

This article is part of Topical Collection on Gene Expression

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary materials S1

All the primers used in this study, including eight pairs of primers for quantitative real-time PCR and twenty-three pairs of primers for precursor sequences of G. biloba. (XLS 22 kb)

Supplementary materials S2

The file shows the information of known miRNAs in female and male leaves of G. biloba, including family members, mature sequence, length, amount of expression, as well as homology analysis. (XLS 73 kb)

Supplementary materials S3

Fold-back structures for known miRNA from the mature leaves of G. biloba. Precursor sequences for known miRNAs were shown in black letters with miRNA sequences highlighted in red. Precursor secondary structures and MEF value were produced using the MFOLD software. (DOC 441 kb)

Supplementary materials S4

(a) The pre-miRNA sequences by PCR amplification and sequencing. (b) The real-time quantitative PCR gel of 8 selected miRNAs. The numbers 1 and 2 showed that the bands amplified using cDNAs as templates obtained from mature female and male leaves, respectively. M denoted markers. (DOC 1875 kb)

Supplementary materials S5

The file shows the information of novel miRNAs in female and male leaves of G. biloba, including mature miRNA sequence, length of precursor (LP), minimal folding free energy (MFE) and transcripts per quarter million (TPM). (XLS 38 kb)

Supplementary materials S6

Fold-back structures for novel miRNA from the mature leaves of G. biloba. Precursor sequences for novel miRNAs were shown in black letters with miRNA sequences highlighted in red. Precursor secondary structures and MEF value were produced using the MFOLD software. (DOC 192 kb)

Supplementary materials S7

Analysis of miRNAs nucleotide bias in two libraries (a) Analysis of first nucleotide bias of miRNAs in the female leaves. (b) Analysis of first nucleotide bias of miRNAs in the male leaves. (c) Analysis of each nucleotide bias of miRNAs in female leaves. (d) Analysis of each nucleotide bias of miRNAs in male leaves. Red A, blue U, brown G, green C. (DOC 503 kb)

Supplementary materials S8

List of the top 20 KEGG pathways in female and male leaves. (XLS 178 kb)

Supplementary materials S9

The 24 known miRNA targets and their putative functions. (XLS 29 kb)

Supplementary materials S10

The 13 novel miRNA targets and their putative functions. (XLS 26 kb)

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Wang, L., Zhao, J., Zhang, M. et al. Identification and characterization of microRNA expression in Ginkgo biloba L. leaves. Tree Genetics & Genomes 11, 76 (2015). https://doi.org/10.1007/s11295-015-0897-3

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