Genome-wide identification of the Phaseolus vulgaris sRNAome using small RNA and degradome sequencing

BMC Genomics

Georgina Hernández³  José Luis Reyes¹  Federico Sánchez¹  Ramanjulu Sunkar²  Yong-Fang Li²  Pablo Peláez¹  Luis Pedro Iñiguez³  Damien Formey³ 

[1] Departamento de Biología Molecular de Plantas, Instituto de Biotecnología (UNAM), Av. Universidad 2001, Cuernavaca 62210, Morelos, Mexico;Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, OK, USA;Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México (UNAM), Av. Universidad 1001, Cuernavaca 62210, Morelos, Mexico

关键词: Common bean;    Legumes;    Nodules;    Degradome;    phasiRNAs;    microRNAs;    Phaseolus vulgaris;   

Others  :  1211978
DOI  :  10.1186/s12864-015-1639-5

 received in 2015-01-07, accepted in 2015-05-18,  发布年份 2015

【 摘 要 】

Background

MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events.

Results

We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study.

Conclusions

We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N₂-fixing symbiotic nodules.

【 授权许可】

   
2015 Formey et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Broughton WJ, Hernández G, Blair M, Beebe S, Gepts P, Vanderleyden J. Beans (Phaseolus spp.) – model food legumes. Plant Soil. 2003; 252:55-128.
• [2]Schmutz J, McClean PE, Mamidi S, Wu GA, Cannon SB, Grimwood J et al.. A reference genome for common bean and genome-wide analysis of dual domestications. Nat Genet. 2014; 46:707-13.
• [3]Kalavacharla V, Liu Z, Meyers BC, Thimmapuram J, Melmaiee K. Identification and analysis of common bean (Phaseolus vulgaris L.) transcriptomes by massively parallel pyrosequencing. BMC Plant Biol. 2011; 11:135.
• [4]Liu Z, Crampton M, Todd A, Kalavacharla V. Identification of expressed resistance gene-like sequences by data mining in 454-derived transcriptomic sequences of common bean (Phaseolus vulgaris L.). BMC Plant Biol. 2012; 12:42.
• [5]Hernández G, Valdés-López O, Ramírez M, Goffard N, Weiller G, Aparicio-Fabre R et al.. Global changes in the transcript and metabolic profiles during symbiotic nitrogen fixation in phosphorus-stressed common bean plants. Plant Physiol. 2009; 151:1221-38.
• [6]Hiz MC, Canher B, Niron H, Turet M. Transcriptome analysis of salt tolerant common bean (Phaseolus vulgaris L.) under saline conditions. PLoS One. 2014; 9:e92598.
• [7]Hernández G, Ramírez M, Valdés-López O, Tesfaye M, Graham MA, Czechowski T et al.. Phosphorus stress in common bean: root transcript and metabolic responses. Plant Physiol. 2007; 144:752-67.
• [8]Valdés-López O, Yang SS, Aparicio-Fabre R, Graham PH, Reyes JL, Vance CP et al.. MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity. New Phytol. 2010; 187:805-18.
• [9]Ramírez M, Flores-Pacheco G, Reyes JL, Luzlvarez A, Drevon JJ, Girard L et al.. Two Common Bean Genotypes with Contrasting Response to Phosphorus Deficiency Show Variations in the microRNA 399-Mediated PvPHO2 Regulation within the PvPHR1 Signaling Pathway. Int J Mol Sci. 2013; 14:8328-44.
• [10]Naya L, Paul S, Valdés-López O, Mendoza-Soto AB, Nova-Franco B, Sosa-Valencia G et al.. Regulation of copper homeostasis and biotic interactions by microRNA 398b in common bean. PLoS One. 2014; 9:e84416.
• [11]Contreras-Cubas C, Rabanal FA, Arenas-Huertero C, Ortiz MA, Covarrubias AA, Reyes JL. The Phaseolus vulgaris miR159a precursor encodes a second differentially expressed microRNA. Plant Mol Biol. 2012; 80:103-15.
• [12]Jones-Rhoades MW, Bartel DP, Bartel B. MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol. 2006; 57:19-53.
• [13]Li S-C, Chan W-C, Hu L-Y, Lai C-H, Hsu C-N, Lin W. Identification of homologous microRNAs in 56 animal genomes. Genomics. 2010; 96:1-9.
• [14]Lin W-C, Li S-C, Lin W-C, Shin J-W, Hu S-N, Yu X-M et al.. Identification of microRNA in the protist Trichomonas vaginalis. Genomics. 2009; 93:487-93.
• [15]Zhang Y-Q, Chen D-L, Tian H-F, Zhang B-H, Wen J-F. Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia. Comput Biol Chem. 2009; 33:391-6.
• [16]Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell. 2009; 136:669-87.
• [17]Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004; 116:281-97.
• [18]Combier J-P, Frugier F, de Billy FF, Boualem A, El-Yahyaoui F, Moreau S et al.. MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes Dev. 2006; 20:3084-8.
• [19]Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CL. Regulation of phosphate homeostasis by microRNA in Arabidopsis. Plant Cell. 2006; 18:412-21.
• [20]Meng Y, Ma X, Chen D, Wu P, Chen M. MicroRNA-mediated signaling involved in plant root development. Biochem Biophys Res Commun. 2010; 393:345-9.
• [21]Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M et al.. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science (80). 2006; 312:436-9.
• [22]Staiger D, Korneli C, Lummer M, Navarro L. Emerging role for RNA-based regulation in plant immunity. New Phytol. 2013; 197:394-404.
• [23]Eckardt NA. The plant cell reviews aspects of microRNA and PhasiRNA regulatory function. Plant Cell. 2013; 25:2382.
• [24]Allen E, Xie ZX, Gustafson AM, Carrington JC. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell. 2005; 121:207-21.
• [25]Vazquez F, Vaucheret H, Rajagopalan R, Lepers C, Gasciolli V, Mallory AC et al.. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell. 2004; 16:69-79.
• [26]Song X, Li P, Zhai J, Zhou M, Ma L, Liu B et al.. Roles of DCL4 and DCL3b in rice phased small RNA biogenesis. Plant J. 2012; 69:462-74.
• [27]Fei Q, Xia R, Meyers BC. Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell. 2013; 25:2400-15.
• [28]Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014; 42(Database issue):D68-73.
• [29]Peláez P, Trejo MS, Iñiguez LP, Estrada-Navarrete G, Covarrubias AA, Reyes JL et al.. Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing. BMC Genomics. 2012; 13:83.
• [30]Han J, Xie H, Kong ML, Sun QP, Li RZ, Pan JB. Computational identification of miRNAs and their targets in Phaseolus vulgaris. Genet Mol Res. 2014; 13:310-22.
• [31]Arenas-Huertero C, Pérez B, Rabanal F, Blanco-Melo D, De la Rosa C, Estrada-Navarrete G et al.. Conserved and novel miRNAs in the legume Phaseolus vulgaris in response to stress. Plant Mol Biol. 2009; 70:385-401.
• [32]Ormeño-Orrillo E, Menna P, Almeida LGP, Ollero FJ, Nicolás MF, Pains Rodrigues E et al.. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.). BMC Genomics. 2012; 13:735.
• [33]Yang X, Li L. miRDeep-P: a computational tool for analyzing the microRNA transcriptome in plants. Bioinformatics. 2011; 27:2614-5.
• [34]Morin RD, O’Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu A-L et al.. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res. 2008; 18:610-21.
• [35]Jones-Rhoades MW. Conservation and divergence in plant microRNAs. Plant Mol Biol. 2012; 80:3-16.
• [36]Li H, Deng Y, Wu T, Subramanian S, Yu O. Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation. Plant Physiol. 2010; 153:1759-70.
• [37]Bustos-Sanmamed P, Bazin J, Hartmann C, Crespi M, Lelandais-Briere C, Lelandais-Brière C. Small RNA pathways and diversity in model legumes: lessons from genomics. Front Plant Sci. 2013; 4:236.
• [38]Zhu Q-H, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F et al.. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res. 2008; 18:1456-65.
• [39]Lertpanyasampatha M, Gao L, Kongsawadworakul P, Viboonjun U, Chrestin H, Liu R et al.. Genome-wide analysis of microRNAs in rubber tree (Hevea brasiliensis L.) using high-throughput sequencing. Planta. 2012; 236:437-45.
• [40]Xia R, Meyers BC, Liu ZZ, Beers EP, Ye S, Liu ZZ. MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA Biogenesis in Eudicots. Plant Cell. 2013; 25:1555-72.
• [41]Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS et al.. High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One. 2007; 2:e219.
• [42]Cuperus JT, Fahlgren N, Carrington JC. Evolution and functional diversification of MIRNA genes. Plant Cell. 2011; 23:431-42.
• [43]Turner M, Yu O, Subramanian S. Genome organization and characteristics of soybean microRNAs. BMC Genomics. 2012; 13:169.
• [44]Formey D, Sallet E, Lelandais-Briere C, Ben CC, Bustos-Sanmamed P, Niebel A et al.. The small RNA diversity from Medicago truncatula roots under biotic interactions evidences the environmental plasticity of the miRNAome. Genome Biol. 2014; 15:457.
• [45]O’Rourke JA, Iniguez LP, Fu F, Bucciarelli B, Miller SS, Jackson SA et al.. An RNA-Seq based gene expression atlas of the common bean. BMC Genomics. 2014; 15:866.
• [46]Addo-Quaye C, Snyder JA, Park YB, Li Y-F, Sunkar R, Axtell MJ. Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome. RNA. 2009; 15:2112-21.
• [47]Meng Y, Gou L, Chen D, Wu P, Chen M. High-throughput degradome sequencing can be used to gain insights into microRNA precursor metabolism. J Exp Bot. 2010; 61:3833-7.
• [48]Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL et al.. Criteria for annotation of plant MicroRNAs. Plant Cell. 2008; 20:3186-90.
• [49]Axtell MJ. Evolution of microRNAs and their targets: are all microRNAs biologically relevant? Biochim Biophys Acta. 2008; 1779:725-34.
• [50]Zhuo Y, Gao G, Shi JA, Zhou X, Wang X. miRNAs: biogenesis, origin and evolution, functions on virus-host interaction. Cell Physiol Biochem. 2013; 32:499-510.
• [51]Colaiacovo M, Lamontanara A, Bernardo L, Alberici R, Crosatti C, Giusti L et al.. On the complexity of miRNA-mediated regulation in plants: novel insights into the genomic organization of plant miRNAs. Biol Direct. 2012; 7:15.
• [52]Axtell MJ, Westholm JO, Lai EC. Vive la différence: biogenesis and evolution of microRNAs in plants and animals. Genome Biol. 2011; 12:221.
• [53]Baskerville S, Bartel DP. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 2005; 11:241-7.
• [54]Elrouby N, Bureau TE. Bs1, a new chimeric gene formed by retrotransposon-mediated exon shuffling in maize. Plant Physiol. 2010; 153:1413-24.
• [55]Ma Z, Coruh C, Axtell MJ. Arabidopsis lyrata small RNAs: transient MIRNA and small interfering RNA loci within the Arabidopsis genus. Plant Cell. 2010; 22:1090-103.
• [56]Li B, Duan H, Li J, Deng XW, Yin W, Xia X. Global identification of miRNAs and targets in Populus euphratica under salt stress. Plant Mol Biol. 2013; 81:525-39.
• [57]Sun F, Guo G, Du J, Guo W, Peng H, Ni Z et al.. Whole-genome discovery of miRNAs and their targets in wheat (Triticum aestivum L.). BMC Plant Biol. 2014; 14:142.
• [58]Xia R, Zhu H, An Y-Q, Beers EP, Liu Z. Apple miRNAs and tasiRNAs with novel regulatory networks. Genome Biol. 2012; 13:R47.
• [59]Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol. 2008; 18:758-62.
• [60]Dai X, Zhao PX. psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res. 2011; 39(Web Server issue):W155-9.
• [61]Meyers BC, Green PJ. Plant MicroRNAs: Methods and Protocols. Hatfield, John M. Walker; 2010.
• [62]Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP. Prediction of plant microRNA targets. Cell. 2002; 110:513-20.
• [63]Brandt R, Cabedo M, Xie Y, Wenkel S. Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment. J Integr Plant Biol. 2014; 56:518-26.
• [64]Boualem A, Laporte P, Jovanovic M, Laffont C, Plet J, Combier J-P et al.. MicroRNA166 controls root and nodule development in Medicago truncatula. Plant J. 2008; 54:876-87.
• [65]Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9:559.
• [66]Valdés-López O, Arenas-Huertero C, Ramírez M, Girard L, Sánchez F, Vance CP et al.. Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots. Plant Cell Environ. 2008; 31:1834-43.
• [67]Liang G, He H, Yu D. Identification of nitrogen starvation-responsive microRNAs in Arabidopsis thaliana. PLoS One. 2012; 7:e48951.
• [68]Olivera M, Tejera N, Iribarne C, Ocaña A, Lluch C. Effect of phosphorous on nodulation and nitrogen fixation by Phaseolus vulgaris. In: First Int Meet Microb Phosphate Solubilization. Velázquez E, Rodríguez-Barrueco C, editors. Springer Netherlands, Dordrecht; 2007: p.157-60. Developments in Plant and Soil Sciences
• [69]Awonaike KO, Lea PJ, Day JM, Roughley RJ, Miflin BJ. Effects of Combined Nitrogen on Nodulation and Growth of Phaseolus vulgaris. Exp Agric. 2008; 16:303.
• [70]Bari R, Pant BD, Stitt M, Scheible W-R. PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol. 2006; 141:988-99.
• [71]Zhai J, Jeong D-H, De Paoli E, Park S, Rosen BD, Li Y et al.. MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes Dev. 2011; 25:2540-53.
• [72]Brocker C, Vasiliou M, Carpenter S, Carpenter C, Zhang Y, Wang X et al.. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics. Planta. 2013; 237:189-210.
• [73]Yoshida S, Kameoka H, Tempo M, Akiyama K, Umehara M, Yamaguchi S et al.. The D3 F-box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis. New Phytol. 2012; 196:1208-16.
• [74]Takahara M, Magori S, Soyano T, Okamoto S, Yoshida C, Yano K et al.. Too much love, a novel Kelch repeat-containing F-box protein, functions in the long-distance regulation of the legume-Rhizobium symbiosis. Plant Cell Physiol. 2013; 54:433-47.
• [75]Arikit S, Xia R, Kakrana A, Huang K, Zhai J, Yan Z et al.. An atlas of soybean small RNAs identifies phased siRNAs from hundreds of coding genes. Plant Cell. 2014; 26:4584-601.
• [76]Wang S, Sun X, Hoshino Y, Yu Y, Jia B, Sun Z et al.. MicroRNA319 positively regulates cold tolerance by targeting OsPCF6 and OsTCP21 in rice (Oryza sativa L.). PLoS One. 2014; 9:e91357.
• [77]Zhou M, Luo H. Role of microRNA319 in creeping bentgrass salinity and drought stress response. Plant Signal Behav. 2014;9.
• [78]Schommer C, Bresso EG, Spinelli SV, Palatnik JF. Role of MicroRNA miR319 in Plant Development. In: MicroRNAs Plant Dev Stress Responses. Sunkar R, editor. Springer Berlin Heidelberg, Berlin, Heidelberg; 2012: p.29-47. Signaling and Communication in Plants
• [79]De Luis A, Markmann K, Cognat V, Holt DB, Charpentier M, Parniske M et al.. Two microRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus. Plant Physiol. 2012; 160:2137-54.
• [80]Ariel F, Brault-Hernandez M, Laffont C, Huault E, Brault M, Plet J et al.. Two direct targets of cytokinin signaling regulate symbiotic nodulation in Medicago truncatula. Plant Cell. 2012; 24:3838-52.
• [81]Lauressergues D, Delaux P-MM, Formey D, Lelandais-Brière C, Fort S, Cottaz S et al.. The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2. Plant J. 2012; 72:512-22.
• [82]Yan Z, Hossain MS, Wang J, Valdés-López O, Liang Y, Libault M et al.. miR172 regulates soybean nodulation. Mol Plant Microbe Interact. 2013; 26:1371-7.
• [83]Nova-Franco B, Íñiguez LP, Valdés-López O, Alvarado-Affantranger X, Leija A, Fuentes SI et al.. The miR172c-AP2-1 Node as a Key Regulator of the Common Bean - Rhizobia Nitrogen Fixation Symbiosis. Plant Physiol. 2015; 114:255547.
• [84]Wang Y, Wang L, Zou Y, Chen L, Cai Z, Zhang S et al.. Soybean miR172c Targets the Repressive AP2 Transcription Factor NNC1 to Activate ENOD40 Expression and Regulate Nodule Initiation. Plant Cell. 2014; 26:4782-801.
• [85]Zhang S, Wang Y, Li K, Zou Y, Chen L, Li X. Identification of Cold-Responsive miRNAs and Their Target Genes in Nitrogen-Fixing Nodules of Soybean. Int J Mol Sci. 2014; 15:13596-614.
• [86]Oldroyd GED, Downie JA. Calcium, kinases and nodulation signalling in legumes. Nat Rev Mol Cell Biol. 2004; 5:566-76.
• [87]Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL et al.. Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell. 2008; 133:128-41.
• [88]Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA et al.. Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting. Plant Cell. 2007; 19:926-42.
• [89]Visser M, van der Walt AP, Maree HJ, Rees DJG, Burger JT. Extending the sRNAome of apple by next-generation sequencing. PLoS One. 2014; 9:e95782.
• [90]Liu Y, Wang Y, Zhu Q-H, Fan L. Identification of phasiRNAs in wild rice (Oryza rufipogon). Plant Signal Behav. 2013;8.
• [91]Manavella PA, Koenig D, Weigel D. Plant secondary siRNA production determined by microRNA-duplex structure. Proc Natl Acad Sci U S A. 2012; 109:2461-6.
• [92]Liu H, Wang X, Zhang H, Yang Y, Ge X, Song F. A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress. Gene. 2008; 420:57-65.
• [93]De Paoli E, Dorantes-Acosta A, Zhai J, Accerbi M, Jeong D-H, Park S et al.. Distinct extremely abundant siRNAs associated with cosuppression in petunia. RNA. 2009; 15:1965-70.
• [94]Xie Q, Frugis G, Colgan D, Chua NH. Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev. 2000; 14:3024-36.
• [95]Broughton WJ, Dilworth MJ. Control of leghaemoglobin synthesis in snake beans. 1971.
• [96]German MA, Luo S, Schroth G, Meyers BC, Green PJ. Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Nat Protoc. 2009; 4:356-62.
• [97]Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. NCBI BLAST: a better web interface. Nucleic Acids Res. 2008; 36:W5-9.
• [98]Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006; 22:1658-9.
• [99]Addo-Quaye C, Miller W, Axtell MJ. CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics. 2009; 25:130-1.
• [100]Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM et al.. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-9.
• [101]Zhi-Liang H, Bao J, Reecy J. CateGOrizer: A Web-Based Program to Batch Analyze Gene Ontology Classification Categories. Online J Bioinforma. 2008; 9:108-12.
• [102]Edwards AWF. Cogwheels of the Mind: The Story of Venn Diagrams. JHU Press, JHU; 2004.
• [103]R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; 2009.
• [104]Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D et al.. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13:2498-504.
• [105]Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10:R25.
• [106]Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3.