Plant Molecular Biology

, Volume 82, Issue 1–2, pp 193–204 | Cite as

Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families

  • Choun-Sea LinEmail author
  • Jeremy J. W. Chen
  • Yao-Ting Huang
  • Chen-Tran Hsu
  • Hsiang-Chia Lu
  • Ming-Lun Chou
  • Li-Chi Chen
  • Chia-I Ou
  • Der-Chih Liao
  • Ysuan-Yu Yeh
  • Song-Bing Chang
  • Su-Chen Shen
  • Fu-Huei Wu
  • Ming-Che Shih
  • Ming-Tsair ChanEmail author


The orchid Erycina pusilla has a short life cycle and relatively low chromosome number, making it a potential model plant for orchid functional genomics. To that end, small RNAs (sRNAs) from different developmental stages of different organs were sequenced. In this miRNA mix, 33 annotated miRNA families and 110 putative miRNA-targeted transcripts were identified in E. pusilla. Fifteen E. pusilla miRNA target genes were found to be similar to those in other species. There were putative novel miRNAs identified by 3 different strategies. The genomic sequences of the four miRNAs that were identified using rice genome as the reference can form the stem loop structure. The t0000354 miRNA, identified using rice genome sequences and a Phalaenopsis study, had a high read count. The target gene of this miRNA is MADS (unigene30603), which belongs to the AP3-PI subfamily. The most abundant miRNA was E. pusilla miR156 (epu-miR156), orthologs of which work to maintain the vegetative phase by repressing the expression of the SQUAMOSA promoter-binding-like (SPL) transcription factors. Fifteen genes in the E. pusilla SPL (EpSPL) family were identified, nine of which contained the putative epu-miR156 target site. Target genes of epu-miR172, also a key regulator of developmental changes in the APETALA2 (EpAP2) family, were identified. Experiments using 5′RLM-RACE demonstrated that the genes EpSPL1, 2, 3, 4, 7, 9, 10, 14 and EpAP2-9, -10, -11 were regulated by epu-miR156 and epu-miR172, respectively.


Orchid  Micro RNA  Transcriptome  Phase transition  sRNA library  AP2 family  SPL family 



We would like to thank Ms. Anita K. Snyder and Ms. Miranda Loney for giving comments on the manuscript. This research was supported by Academia Sinica and Development Program of Industrialization for Agricultural Biotechnology, Taiwan.

Supplementary material

11103_2013_55_MOESM1_ESM.pptx (85 kb)
Primer sequences used in this report (PPTX 84 kb)
11103_2013_55_MOESM2_ESM.pptx (67 kb)
Putative small RNA synthesis pathway genes in Erycina pusilla (PPTX 66 kb)
11103_2013_55_MOESM3_ESM.rar (85.5 mb)
Sequences and read count information of epu-miRNA families (RAR 87509 kb)
11103_2013_55_MOESM4_ESM.txt (22 kb)
Conserved miRNA in E. pusilla (TXT 22 kb)
11103_2013_55_MOESM5_ESM.xlsx (24 kb)
Putative target genes of conserved miRNA in E. pusilla (XLSX 24 kb)
11103_2013_55_MOESM6_ESM.txt (1.6 mb)
The putative novel miRNA which were seleted by tag2miRNA (TXT 1656 kb)
11103_2013_55_MOESM7_ESM.xlsx (363 kb)
The putative novel miRNA (20-22 bps) which were selected by tag2miRNA (XLSX 363 kb)
11103_2013_55_MOESM8_ESM.txt (5.7 mb)
Putative target genes of novel miRNA in Supplemental file 7 (TXT 5842 kb)
11103_2013_55_MOESM9_ESM.xlsx (10 kb)
The putative novel miRNA identified by comparison to rice genome (XLSX 9 kb)
11103_2013_55_MOESM10_ESM.txt (4 kb)
Putative target genes of the novel miRNA in Supplemental file 9 (TXT 4 kb)
11103_2013_55_MOESM11_ESM.txt (1 kb)
Putative novel orchid miRNA identified by comparison to Phalaenopsis (TXT 0 kb)
11103_2013_55_MOESM12_ESM.txt (3 kb)
Putative target genes of the novel orchid miRNA in Supplemental file 11 (TXT 3 kb)
11103_2013_55_MOESM13_ESM.pptx (80 kb)
Supplementary material 13 (PPTX 79 kb)
11103_2013_55_MOESM14_ESM.pptx (81 kb)
Supplementary material 14 (PPTX 81 kb)
11103_2013_55_MOESM15_ESM.bmp (4.6 mb)
Subcellular localization of EpSPL fused with fluorescent proteins. The plasmids containing fusions of GFP and EpSPL1, EpSPL4, or EpSPL9 driven by the 35S promoter were transiently expressed in orchid petal (Hsu et al., 2011), while fusion between YFP and EpSPL2 or EpSPL13 were delivered by particle bombardment (upper left panels). The NLS domain of VirD2 fused with mCherry was used to mark the nucleus (upper right panels). The corresponding bright field images (lower left panels) are overlaid with the two fluorescent images (lower right panels). The lower right panel set shows the transformation efficiency of EpSPL2 (BMP 4699 kb)
11103_2013_55_MOESM16_ESM.bmp (2.2 mb)
Phylogenetic analysis of SPL proteins based on the amino acid sequences of the SBP domains.The phylogenetic tree was generated by the neighbor-joining (NJ) algorithm using Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (Tamura et al., 2007). The numbers on major branches indicate bootstrap percentages for 1,000 replicate analyses. All sequences were collected from the GenBank database following Xie, et al. (2006). The SPL genes containing putative complementary sequences to miR156 are underlined in red. The SPL genes from E. pusilla are underlined in black. Subgroups are numbered S1-S6 and are highlighted by color panels (BMP 2210 kb)
11103_2013_55_MOESM17_ESM.pptx (104 kb)
Tissue-specific expression patterns of EpSPL genes. RNA was extracted from five different tissues of E. pusilla and subjected to cDNA synthesis: R: roots; L: leaves, P: peduncle; F: flowers, C: capsule, PC: protocorn. The quantitative RT-PCR was undertaken using the primers in Table S1. The E. pusilla actin gene was used as the internal control (PPTX 104 kb)
11103_2013_55_MOESM18_ESM.bmp (2.5 mb)
Phylogenetic analysis of the AP2 subfamily. The phylogenetic tree was generated by the neighbor-joining (NJ) algorithm using Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (Tamura et al., 2007). The numbers on major branches indicate bootstrap percentages for 1,000 replicate analyses. The AP2 genes containing the putative complementary sequence of miR172 are underlined in red, while AP2 genes from E. pusilla are underlined in black. Species names and accession numbers are indicated to the right of each protein name. At, Arabidopsis; Ep, E. pusilla; Os, rice. Subgroups labeled ANT and AP2 are according to Shigyo et al. (2006) (BMP 2580 kb)
11103_2013_55_MOESM19_ESM.pptx (66 kb)
EpAP2 genes identified from the E. pusilla transcriptome database. The Arabidopsis AP2 amino acid sequences were used for tblastn searches against the transcriptome database of E. pusilla. The primers derived from these sequences were used for the BAC clone screening. The identified BAC clones were sequenced by NGS and assembled. Twelve EpAP2 genes were identified. These genes were confirmed by blastx against the Arabidopsis protein database. The highest hit in the Arabidopsis AP2 family is listed (PPTX 66 kb)
11103_2013_55_MOESM20_ESM.pptx (71 kb)
Supplementary material 20 (PPTX 70 kb)
11103_2013_55_MOESM21_ESM.bmp (1.3 mb)
Exon–intron structures of EpAP2 genes. The lines indicate introns and rectangles indicate exons (BMP 1367 kb)
11103_2013_55_MOESM22_ESM.bmp (2.6 mb)
Subcellular localization of EpAP2 fusion with fluorescent proteins. Plasmids harboring a GFP fusion with EpAP2-9 (left), EpAP2-10 (central), or EpAP2-12 (right) driven by the 35S promoter were transiently expressed in orchid petal (Hsu et al., 2011; upper left panels). These plasmids were delivered by the particle bombardment method. The NLS domain of VirD2 fused with mCherry was used as the nuclear marker (upper right panels). Bright field (lower left panels) and overlays (Lower right panels) are shown (BMP 2667 kb)
11103_2013_55_MOESM23_ESM.pptx (93 kb)
Tissue-specific expression patterns of EpAP2 genes. RNA was extracted from five different tissues of E. pusilla and subjected to cDNA synthesis: R: roots; L: leaves, P: peduncles; F: flowers, C: capsule, PC: protocorn. The quantitative RT-PCR was conducted using the primers in Table S1 (PPTX 92 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Choun-Sea Lin
    • 1
    Email author
  • Jeremy J. W. Chen
    • 2
  • Yao-Ting Huang
    • 3
  • Chen-Tran Hsu
    • 1
  • Hsiang-Chia Lu
    • 4
  • Ming-Lun Chou
    • 5
  • Li-Chi Chen
    • 2
  • Chia-I Ou
    • 3
  • Der-Chih Liao
    • 1
  • Ysuan-Yu Yeh
    • 6
  • Song-Bing Chang
    • 6
  • Su-Chen Shen
    • 7
  • Fu-Huei Wu
    • 1
  • Ming-Che Shih
    • 1
  • Ming-Tsair Chan
    • 1
    • 4
    Email author
  1. 1.Agricultural Biotechnology Research CenterAcademia SinicaTaipeiTaiwan
  2. 2.Institute of Biomedical SciencesNational Chung-Hsing UniversityTaichungTaiwan
  3. 3.Department of Computer Science and Information EngineeringNational Chung Cheng UniversityChia-iTaiwan
  4. 4.Biotechnology Center in Southern TaiwanAcademia SinicaTainanTaiwan
  5. 5.Department of Life SciencesTzu Chi UniversityHualienTaiwan
  6. 6.Department of Life SciencesNational Cheng Kung UniversityTainanTaiwan
  7. 7.Scientific Instrument CenterAcademia SinicaTaipeiTaiwan

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