Advertisement

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 Lin
  • 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 Chan
Article

Abstract

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.

Keywords

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

Notes

Acknowledgments

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)

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  2. An FM, Hsiao SR, Chan MT (2011) Sequencing-based approaches reveal low ambient temperature-responsive and tissue-specific microRNAs in phalaenopsis orchid. PLoS ONE 6:e18937PubMedCrossRefGoogle Scholar
  3. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741PubMedCrossRefGoogle Scholar
  4. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208PubMedCrossRefGoogle Scholar
  5. Birkenbihl RP, Jach G, Saedler H, Huijser P (2005) Functional dissection of the plant-specific SBP-domain: overlap of the DNA-binding and nuclear localization domains. J Mol Biol 352:585–596PubMedCrossRefGoogle Scholar
  6. Calderon-Villalobos LIA, Kuhnle C, Dohmann EMN, Li H, Bevan M, Schwechheimer C (2005) The evolutionarily conserved TOUGH protein is required for proper development of Arabidopsis thaliana. Plant Cell 17:2473–2485PubMedCrossRefGoogle Scholar
  7. Chang WC, Lee TY, Huang HD, Huang HY, Pan RL (2008) PlantPAN: plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC Genomics 9:561PubMedCrossRefGoogle Scholar
  8. Chase MW, Hanson L, Albert VA, Whitten WM, Williams NH (2005) Life history evolution and genome size in subtribe Oncidiinae (Orchidaceae). Ann Bot 95:191–199PubMedCrossRefGoogle Scholar
  9. Chen X (2004) A MicroRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025PubMedCrossRefGoogle Scholar
  10. Chen X (2009) Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25:21–44PubMedCrossRefGoogle Scholar
  11. Chi X, Yang Q, Chen X, Wang J, Pan L, Chen M, Yang Z, He Y, Liang X, Yu S (2011) Identification and characterization of microRNAs from peanut (Arachis hypogaea L.) by high-throughput sequencing. PLoS ONE 6:e27530PubMedCrossRefGoogle Scholar
  12. Chiu YT, Lin CS, Chang C (2011) In vitro fruting and seed production in Erycina Pusilla (L.) N. H. Willams and M. W. Chase. Propag Ornam Plants 11:131–136Google Scholar
  13. Chou ML, Shih MC, Chan MT, Liao SY, Hsu CT, Haung YT, Chen JJ, Liao DC, Wu FH, Lin CS (2013) Global transcriptome analysis and identification of a CONSTANS-like gene family in the orchid Erycina pusilla. Planta. doi: 10.1007/s00425-013-1850-z PubMedGoogle Scholar
  14. Chuck G, Cigan AM, Saeteurn K, Hake S (2007) The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nat Genet 39:544–549PubMedCrossRefGoogle Scholar
  15. Cuperus JT, Fahlgren N, Carrington JC (2011) Evolution and functional diversification of MIRNA genes. Plant Cell 23:431–442PubMedCrossRefGoogle Scholar
  16. Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:W155–W159PubMedCrossRefGoogle Scholar
  17. Felix LP, Guerra M (1999) Chromosome analysis in Psygmorchis pusilla (L.) Dodson and Dressler: the smallest chromosome number known in Orchidaceae. Caryologia 52:165–168CrossRefGoogle Scholar
  18. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158PubMedCrossRefGoogle Scholar
  19. Hsu CT, Liao DC, Wu FH, Liu NT, Shen SC, Chou SJ, Tung SY, Yang CH, Chan MT, Lin CS (2011) Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey. BMC Plant Biol 11:60PubMedCrossRefGoogle Scholar
  20. Hunter C, Poethig RS (2003) miSSING LINKS: miRNAs and plant development. Curr Opin Genet Dev 13:372–378PubMedCrossRefGoogle Scholar
  21. Jamalkandi SA, Masoudi-Nejad A (2009) Reconstruction of Arabidopsis thaliana fully integrated small RNA pathway. Funct Integr Genomics 9:419–432PubMedCrossRefGoogle Scholar
  22. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195PubMedCrossRefGoogle Scholar
  23. Kim S, Soltis PS, Wall K, Soltis DE (2006) Phylogeny and domain evolution in the APETALA2-like gene family. Mol Biol Evol 23:107–120PubMedCrossRefGoogle Scholar
  24. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120PubMedCrossRefGoogle Scholar
  25. Klein J, Saedler H, Huijser P (1996) A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA. Mol Gen Genet 250:7–16PubMedGoogle Scholar
  26. Lee DY, Lee J, Moon S, Park SY, An G (2007) The rice heterochronic gene SUPERNUMERARY BRACT regulates the transition from spikelet meristem to floral meristem. Plant J 49:64–78PubMedCrossRefGoogle Scholar
  27. Ling LZ, Zhang SD (2012) Unraveling the distribution and evolution of miR156-targeted SPLs in plants by phylogenetic analysis. Plant Div Res 34:33–46Google Scholar
  28. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056PubMedCrossRefGoogle Scholar
  29. Mathieu J, Yant LJ, Murdter F, Kuttner F, Schmid M (2009) Repression of flowering by the miR172 target SMZ. PLoS Biol 7:e1000148PubMedCrossRefGoogle Scholar
  30. Miura K, Ikeda M, Matsubara A, Song XJ, Ito M, Asano K, Matsuoka M, Kitano H, Ashikari M (2010) OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat Genet 42:545–549PubMedCrossRefGoogle Scholar
  31. Mulder NJ, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bradley P, Bork P, Bucher P, Cerutti L, Copley R, Courcelle E, Das U, Durbin R, Fleischmann W, Gough J, Haft D, Harte N, Hulo N, Kahn D, Kanapin A, Krestyaninova M, Lonsdale D, Lopez R, Letunic I, Madera M, Maslen J, McDowall J, Mitchell A, Nikolskaya AN, Orchard S, Pagni M, Ponting CP, Quevillon E, Selengut J, Sigrist CJ, Silventoinen V, Studholme DJ, Vaughan R, Wu CH (2005) InterPro, progress and status in 2005. Nucleic Acids Res 33:D201–D205PubMedCrossRefGoogle Scholar
  32. Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140:411–432PubMedCrossRefGoogle Scholar
  33. Pan IC, Liao DC, Wu FH, Daniell H, Singh ND, Chang C, Shih MC, Chan MT, Lin CS (2012) Complete chloroplast genome sequence of an orchid model plant candidate: Erycina pusilla apply in tropical Oncidium breeding. PLoS ONE 7:e34738PubMedCrossRefGoogle Scholar
  34. Pelaez P, Trejo MS, Iniguez LP, Estrada-Navarrete G, Covarrubias AA, Reyes JL, Sanchez F (2012) Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing. BMC Genomics 13:83PubMedCrossRefGoogle Scholar
  35. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110PubMedCrossRefGoogle Scholar
  36. Shigyo M, Ito M (2004) Analysis of gymnosperm two-AP2-domain-containing genes. Dev Genes Evol 214:105–114PubMedCrossRefGoogle Scholar
  37. Shigyo M, Hasebe M, Ito M (2006) Molecular evolution of the AP2 subfamily. Gene 366:256–265PubMedCrossRefGoogle Scholar
  38. Su CL, Chao YT, Yen SH, Chen CY, Chen WC, Chang YC, Shih MC (2013) Orchidstra: an integrated orchid functional genomics database. Plant Cell Physiol 54:e11PubMedCrossRefGoogle Scholar
  39. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  40. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  41. Unte US, Sorensen AM, Pesaresi P, Gandikota M, Leister D, Saedler H, Huijser P (2003) SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis. Plant Cell 15:1009–1019PubMedCrossRefGoogle Scholar
  42. Varkonyi-Gasic E, Lough RH, Moss SM, Wu R, Hellens RP (2012) Kiwifruit floral gene APETALA2 is alternatively spliced and accumulates in aberrant indeterminate flowers in the absence of miR172. Plant Mol Biol 78:417–429PubMedCrossRefGoogle Scholar
  43. Wu G, Poethig RS (2006) Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133:3539–3547PubMedCrossRefGoogle Scholar
  44. Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138:750–759PubMedCrossRefGoogle Scholar
  45. Xie K, Wu C, Xiong L (2006) Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiol 142:280–293PubMedCrossRefGoogle Scholar
  46. Zhang B, Pan X, Cobb GP, Anderson TA (2006) Plant microRNA, a small regulatory molecule with big impact. Dev Biol 289:3–16PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Choun-Sea Lin
    • 1
  • 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
  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

Personalised recommendations