The application of DNA sequencing technologies has a major impact on molecular biology, especially in understanding genes interaction in a certain condition. Due to a large number of genes produced by this high-throughput technology, a proper analysis tool is needed for data interpretation. ClueGO is a bioinformatics tool, an easy to use Cytoscape plug-in that strongly improves biological function interpretation of genes. It analyzes a cluster or comparing two clusters and comprehensively visualizes their group functions. This tool is applied to identify biological networks of genes involved in embryogenesis of oil palm, the most critical phase in oil palm tissue culture process. Two ESTs sequencing data from the GenBank database under accession number EY396120-EY413718 and DW247764-DW248770 were used in this study. Fifty-two and one hundred eight groups of genes were identified using biological process in Gene Ontology setting from the database of EY396120-EY413718 and DW247764-DW248770, respectively. Thirty-one groups of genes were consistently occurred in both ESTs. According to the literature, these genes play an important role in cell formations and developments, stresses and stimulus responses, photosynthesis and metabolic processes that indicate the involvement of these groups of genes in oil palm embryogenesis processes. ClueGO is the appropriate tool to analyze a large data set of genes in a specific condition, such as embryogenesis of oil palm. Keywords: callus embryogenesis; Cytoscape plug-in; DNA sequencing; expressed sequence tag; KEGG pathway

Achmad Tanjung, Zulfikar

Liwang, Tony

Sudania, Widyartini Made

Toruan-Mathius, Nurita

English

KnE Publishing Platform

ICBS Conference ProceedingsInternational Conference on Biological Science (2015),Volume 2017Conference PaperBiological Network Analysis of Genes Involvein Embryogenesis of Oil Palm using ClueGOWidyartini Made Sudania, Zulfikar Achmad Tanjung, Nurita Toruan-Mathius,and Tony LiwangPlant Production and Biotechnology Division, PT. SMART Tbk, Sinar Mas Land Plaza, 2nd Tower,10th Fl., Jalan M.H. Thamrin No.51, Jakarta 10350, IndonesiaAbstractThe application of DNA sequencing technologies has a major impact on molecularbiology, especially in understanding genes interaction in a certain condition. Due to alarge number of genes produced by this high-throughput technology, a proper analysistool is needed for data interpretation. ClueGO is a bioinformatics tool, an easy to useCytoscape plug-in that strongly improves biological function interpretation of genes.It analyzes a cluster or comparing two clusters and comprehensively visualizes theirgroup functions. This tool is applied to identify biological networks of genes involvedin embryogenesis of oil palm, the most critical phase in oil palm tissue culture process.Two ESTs sequencing data from the GenBank database under accession numberEY396120-EY413718 and DW247764-DW248770 were used in this study. Fifty-twoand one hundred eight groups of genes were identified using biological process inGene Ontology setting from the database of EY396120-EY413718 and DW247764-DW248770, respectively. Thirty-one groups of genes were consistently occurred inboth ESTs. According to the literature, these genes play an important role in cellformations and developments, stresses and stimulus responses, photosynthesis andmetabolic processes that indicate the involvement of these groups of genes in oilpalm embryogenesis processes. ClueGO is the appropriate tool to analyze a large dataset of genes in a specific condition, such as embryogenesis of oil palm.Keywords: callus embryogenesis; Cytoscape plug-in; DNA sequencing; expressedsequence tag; KEGG pathways.1. IntroductionThe use of clonal planting materials in large-scale oil palm plantations offers severaladvantages compared with seedling planting materials. It allows high productivity(increasing yields between 20% to 30%), rapid multiplication and uniform growth ofpalmswith desired traits [1]. As the limitation, it has various critical aspects that requireextra precaution for successful inductions and developments of somatic embryos suchas genotype, cytoskeleton, arabinogalactan protein, environment and plant hormones[2]. Somatic embryo formation is an important and major constraint in oil palm tissueculture due to low average rate of embryogenesis at 3% to 6% [3]. On the other hand,How to cite this article: Widyartini Made Sudania, Zulfikar Achmad Tanjung, Nurita Toruan-Mathius, and Tony Liwang, (2017), “Biological NetworkAnalysis of Genes Involve in Embryogenesis of Oil Palm using ClueGO” in ICBS Conference Proceedings, International Conference on Biological Science(2015), KnE Life Sciences, pages 265–274. DOI 10.18502/kls.v3i4.714Page 265Corresponding Author:Widyartini Made Sudaniabiotechnology@sinarmas-agri.comReceived: 11 February 2017Accepted: 08 March 2017Published: 26 March 2017Publishing services providedby Knowledge EWidyartini Made Sudaniaet al. This article is distributedunder the terms of theCreative Commons AttributionLicense, which permitsunrestricted use andredistribution provided thatthe original author and sourceare credited.Selection and Peer-reviewunder the responsibility of theICBS Conference Committee.ICBS Conference ProceedingsFigure 1: Flowchart of biological network analysis using ClueGO software.information on molecular mechanisms and genes associated with embryogenesis pro-cesses in oil palm tissue culture remains limited.Numerous approaches have been used to understand the complexity of gene inter-action in embryogenesis of oil palm tissue culture, including expressed sequence tags(EST). Some of EST data sequences have been published in GenBank database [4, 5].These data can be analyzed in advance to get more comprehensive information aboutbiological networks of genes, which are relevant in embryogenesis using bioinformat-ics tools, such as ClueGO.ClueGO is a plug-in Cytoscape software that strongly improves biological interpreta-tions of genes. ClueGO integrates Gene Ontology (GO) terms as well as KEGG/BioCartapathways and creates a functionally organized GO/pathway term networks [6]. As theClueGO developer [6], has introduced and conducted an experimental trial of ClueGOfor biological networks analysis in 2009 to analyze natural (NK) cell genes in human.It showed a good result in grouping either up-regulated or down-regulated genesinto some biological networks. Based on this result, this tool was applied to identifybiological networks of genes from EST sequencing data during embryogenesis of oilpalm callus.DOI 10.18502/kls.v3i4.714 Page 266ICBS Conference Proceedings Figure 2: Hypothesized molecular and physiological pathways of somatic embryogenesis. Source: Elhitiet al. [10].2. Materials and MethodsThis study utilized two oil palm EST libraries from GenBank under accession numbersof EY396120-EY413718 and DW247764-DW248770 [4, 5]. There were two librariesunder accession of EY396120-EY413718, namely embryogenic callus (EC) and non-embryogenic callus (NEC), and one library under accession numbers EY396120-EY413718, which was embryoid. The analysis comprised of sequence assembly usingcap3 [7], sequence blasting (similarity analysis) using BLASTX software ver.2.2.25[8], gene annotating using BLAST2GO software ver.2.8 [9], and biological networkinganalysis using ClueGO software ver.1.8 [6] (see Figure 1).Hypothesized molecular and physiological pathways of plant somatic embryogene-sis described by Elhiti et al. [10] were used as a reference library to identify all stagesin embryogenesis that were affected by these genes product shown in Figure 2.3. Result and DiscussionThere were 53 and 108 biological network groups, which putatively involved in oil palmembryogenesis were identified using ClueGo analysis from EST EY396120-EY413718DOI 10.18502/kls.v3i4.714 Page 267ICBS Conference ProceedingsFigure 3: Biological network groups of genes derived from EST EY396120-EY413718.and DW247764-DW248770, respectively (see Figure 3 and Figure 4). Biological net-work groups were illustrated as nodes, linked based on their kappa score levels (≥0.5).The node size (small to big) and color gradient (red to dark red) were representedmoresignificance.Specific genes networking overviews and their distributions in EC from ESTEY396120-EY413718 and EST DW247764-DW248770 showed several groups that con-sisted of a large number of genes (see Figure 5 and Figure 6). These groups of geneswere suspected to be up-regulated during embryogenesis process.There were 31 groups of genes networking, which consistently occurred in the twoEST datasets were identified from the data comparison analysis (Table 1). Severalgroups of genes were up-regulated in the two data sets. These groups of genes mayhave a function in regulating somatics embryogenesis, especially in embryonic induc-tion. According to the literature in plant somatic embryogenesis, eleven functions inDOI 10.18502/kls.v3i4.714 Page 268ICBS Conference ProceedingsFigure 4: Biological network groups of genes derived from EST DW247764-DW24877.somatics embryogenesis were affected by these groups of genes. These functionsare control cell positions and its shape correctly during proliferations; promotions ofcell elongations, cell divisions, and dedifferentiations throughout the plant cell lifecycles; ethylene precursor; meristem formation and regulation; modification in cellwall composition (SE induction effect); ethylene synthesis; Gibberellin (GA) precursor;Jasmonic acid ( JA) precursor; genetic information to control totipotency; and cell cycles[10–16].DOI 10.18502/kls.v3i4.714 Page 269ICBS Conference ProceedingsFigure 5: Genes networking overviews from EST EY396120-EY413718 data set.Figure 6: Genes networking overview from EST DW247764-DW248770 data set.DOI 10.18502/kls.v3i4.714 Page 270ICBS Conference ProceedingsNo Groups of genes Function in embryogenesis Phase1 actin filament bundleassembly [11, 12]control cell position and its shape correctlyduring proliferation; needed indevelopmentalswitch from unpolarized topolarized cells in response to auxindedifferentiation2 actin polymerization ordepolymerization*)[11, 12]control cell position and its shape correctlyduring proliferation(11); needed indevelopmentalswitch from unpolarized topolarized cells in response to auxindedifferentiation3 brassinosteroid-mediatedsignaling pathway [10]promotion of cell elongation, cell division,and dedifferentiation throughout the plantcell life cyclededifferentiation4 carboxylic acid biosyntheticprocess* [13]ethylene precursor (low level inhibited theformation of somatic embryos, whereas highlevels elevated the frequency of somaticembryos induction)dedifferentiation5 carboxylic acid catabolicprocess* [13, 16]ethylene precursor (low level inhibited theformation of somatic embryos, whereas highlevels elevated the frequency of somaticembryos induction)dedifferentiation6 cell tip growth [10] meristem formation and regulation commitment7 cellular polysaccharidebiosynthetic process* [14]modification in cell wall composition (SEinduction effect)commitment8 cellulose metabolic process[14]modification in cell wall composition (SEinduction effect)commitment9 cytokinesis by cell plateformation [11, 12]control cell position and its shape correctlyduring proliferation; needed indevelopmentalswitch from unpolarized topolarized cells in response to auxindedifferentiation10 epidermis development*)[10]meristem formation and regulation commitment11 fruit development* [15] involved in ethylene synthesis dedifferentiation12 gibberellin metabolicprocess*) [10]Gibberellin (GA) precursor (low levelinhibited the formation of somatic embryos,whereas high levels elevated the frequencyof somatic embryos induction)dedifferentiation13 jasmonic acid metabolicprocess [10, 16]Jasmonic acid ( JA) precursor (low levelinhibited the formation of somatic embryos,whereas high levels elevated the frequencyof somatic embryos induction), ethyleneprecursordedifferentiation14 lateral rootdevelopment)[10]meristem formation and regulation commitment15 leaf development [10] meristem formation and regulation commitment16 meristem structuralorganization [10]meristem formation and regulation commitment17 monocarboxylic acidmetabolic process [14]modification in cell wall composition (SEinduction effect)commitment18 negative regulation oftranscription,DNA-templated [10]genetic information required to controltotipotencyexpression oftotipotency19 plant-type cell wallloosening [10]involved in cell cycle commitmentDOI 10.18502/kls.v3i4.714 Page 271ICBS Conference ProceedingsNo Groups of genes Function in embryogenesis Phase20 positive regulation of cellularprotein metabolic process[10]genetic information required to controltotipotencyexpression oftotipotency21 positive regulation oftranscription,DNA-templated [10]genetic information required to controltotipotencyexpression oftotipotency22 protein acylation)[10] genetic information required to controltotipotencyexpression oftotipotency23 protein targeting to vacuole[10]genetic information required to controltotipotencyexpression oftotipotency24 proteolysis*)[10] genetic information required to controltotipotencytotipotency25 regulation of abscisicacid-activated signalingpathway [10, 13, 16]ethylene precursor (low level inhibited theformation of somatic embryos, whereas highlevels elevated the frequency of somaticembryos induction)dedifferentiation26 regulation of proteinphosphorylation*)[10]genetic information required to controltotipotencyexpression oftotipotency27 regulation of RNA metabolicprocess [10]genetic information required to controltotipotencyexpression oftotipotency28 regulation oftransmembrane transporteractivity [10]genetic information required to controltotipotencyexpression oftotipotency29 response to fructose [14] modification in cell wall composition (SEinduction effect)commitment30 seed maturation [10] meristem formation and regulation commitment31 stomatal complexdevelopment [10]meristem formation and regulation commitment*: up-regulated in EST EY396120-EY413718 data set; ): up-regulated in EST DW247764-DW248770;*): up-regulated in the two data setsT 1: List of genes networking groups consistently occurred in the two EST datasets.4. ConclusionThirty-one groups of genes that may play an important role in embryogenesis of oilpalm in vitro culture were identified using ClueGO analysis. These groups affect ineleven functions of embryogenesis based on numerous previous publications in plantsomatic embryogenesis studies. ClueGO is considered as the appropriate bioinformatictool to conduct in silico analysis and identify candidate genes that may impact in aspecific condition, including oil palm embryogenesis from large data set of genes, suchas EST data set.AcknowledgementsThe authors would like to thank PT. SMART Tbk management for making this researchand paper possible.DOI 10.18502/kls.v3i4.714 Page 272ICBS Conference ProceedingsReferences[1] M. B. Wahid, S. N. A. Abdullah, and I. E. Henson, “Oil palm - Achievements andpotential,” Plant Production Science, vol. 8, no. 3, pp. 288–297, 2005.[2] J. Šamaj, M. Bobák, A. Blehová, and A. Pret’Ová, “Importance of cytoskeleton andcell wall in somatic embryogenesis,” Plant Cell Monographs, vol. 2, pp. 35–50, 2005.[3] PBH. Tinker and RHV. Corley, “The Oil Palm,” in The Oil Palm, p. 284, BlackwellScience, Oxford, 4th edition, 2003.[4] E.-T. L. Low, H. Alias, S.-H. Boon et al., “Oil palm (Elaeis guineensis Jacq.) tissueculture ESTs: Identifying genes associated with callogenesis and embryogenesis,”BMC Plant Biology, vol. 8, article no. 62, 2008.[5] P.-L. Chan, L.-S. Ma, E.-T. L. Low et al., “Normalized embryoid cDNA library of oilpalm (Elaeis guineensis),” Electronic Journal of Biotechnology, vol. 13, no. 1, 2010.[6] G. Bindea, B. Mlecnik, H. Hackl et al., “ClueGO: A Cytoscape plug-in to decipher func-tionally grouped gene ontology and pathway annotation networks,” Bioinformatics,vol. 25, no. 8, pp. 1091–1093, 2009.[7] X. Huang and A. Madan, “CAP3: A DNA sequence assembly program,” GenomeResearch, vol. 9, no. 9, pp. 868–877, 1999.[8] S. F. Altschul, W. Gish, W. Miller, E. 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Biological Network Analysis of Genes Involve in Embryogenesis of Oil Palm using ClueGO

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Biological Network Analysis of Genes Involve in Embryogenesis of Oil Palm using ClueGO

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