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Genome-wide mining for microRNAs and their targets in Betula luminifera using high-throughput sequencing and degradome analyses

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Abstract

microRNAs (miRNAs) play vital roles in plants regulating a panoply of biological processes, such as development, hormone signaling, and stress response, by inhibiting target genes at the post-transcriptional level. However, the roles of miRNAs in Betula luminifera remain elusive. To mine for B. luminifera miRNAs and targets, we used a deep sequencing approach to analyze the sRNAs and degradome sequencing of mixed samples, including roots, stems, and leaves. A total of 114 known miRNAs or miRNA*s from 44 families, and 24 novel miRNAs and 17 miRNA*s plus 15 plausible miRNA candidates were identified, of which 36 known miRNAs, 29 miRNA*s, and all novel miRNAs had precursor sequences. Additionally, 49 targets for 19 known miRNA families and seven miRNA*s, and seven targets for novel miRNAs were identified using a high-throughput degradome-sequencing approach. The conserved miRNA targets were mainly transcription factors, whereas the miRNA* targets were mainly protein-coding genes, with preferential propensity to functional enzymes. A Gene Ontology analysis showed that the predicted targets were classified into 62 biological processes, 20 cellular components, and 28 molecular functions, respectively. We found two different targets for miR396a* and miR396c*, and the target changed when the miR156d precursor cleavage site was shifted toward the 5′-end by two nucleotides, indicating the diverse regulatory roles of MIRNA genes. Furthermore, three targets identified by degradome sequencing were validated further through 5′ rapid amplification of cDNA ends. The expression patterns of the randomly selected miRNAs varied among different tissues. miR164 expression was induced under nitrogen starvation, with tissue-specific expression patterns, and was negatively correlated with the NAC1 target gene in roots and leaves, but not in stems. This study is a transcriptome-based analysis of miRNAs and the degradome, providing useful information to explore the functions of miRNAs and their target genes in B. luminifera.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 31300566) and Zhejiang Province Science and Technology Support Program (No. 2012C12908-8).

Author contributions

JZ, MH and ZT conceived and designed the experiments, and wrote the paper. MH, JL, LC and JW performed the experiments, JZ and ZT analyzed the data, YP and LC contributed reagents/materials/ analytical tools. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Huancheng Road, Lin’an, Hangzhou, 311300, Zhejiang, People’s Republic of China

    Junhong Zhang, Menghui Huang, Ying Pan, Longjun Cheng, Jun Wu & Zaikang Tong

  2. Hunan Academy of Forestry, Changsha, 410004, Hunan, People’s Republic of China

    Junsheng Liang

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  1. Junhong Zhang
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  2. Menghui Huang
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  4. Ying Pan
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  5. Longjun Cheng
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  6. Jun Wu
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  7. Zaikang Tong
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Correspondence to Zaikang Tong.

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The authors declare that they have no competing interests.

Data archive statement

The sequencing data is currently being submitted to NCBI’s Gene Expression Omnibus (GEO, accession numbers: GSE80074), and the accession numbers will be supplied once available.

Additional information

Communicated by M. Troggio

Junhong Zhang and Menghui Huang contributed equally to this work.

Electronic supplementary material

Supplementary Table 1

5′ RACE Primers for validation of miRNA targets (DOC 29 kb)

Supplementary Table 2

miRNA primers used in qRT-PCR (DOC 34 kb)

Supplementary Table 3

The miRNA precursor sequences in B. luminifera (XLS 44 kb)

Supplementary Table 4

Identification of known miRNAs ambiguously from B. luminifera (DOC 81 kb)

Supplementary Table 5

Isolation and identification of candidate miRNAs in B. luminifera (DOC 46 kb)

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Zhang, J., Huang, M., Liang, J. et al. Genome-wide mining for microRNAs and their targets in Betula luminifera using high-throughput sequencing and degradome analyses. Tree Genetics & Genomes 12, 99 (2016). https://doi.org/10.1007/s11295-016-1047-2

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  • DOI: https://doi.org/10.1007/s11295-016-1047-2

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