1. bookVolume 61 (2012): Issue 1-6 (December 2012)
Journal Details
First Published
22 Feb 2016
Publication timeframe
1 time per year
access type Open Access

Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

Published Online: 01 Aug 2017
Page range: 280 - 286
Received: 12 Jun 2012
Journal Details
First Published
22 Feb 2016
Publication timeframe
1 time per year

Reference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

Key words

ALVAREZ, C., L. VALLEDOR, R. HASBÚN, M. SANCHEZ-OLATE and D. RÍOS (2011): Variation in gene expression profile with aging of Pinus radiata D. Don. BMC Proceedings 2011, 5 (Suppl 7): P62.Search in Google Scholar

ANDERSEN, C. L., J. L. JENSEN and T. F. ORNTOFT (2004): Normalization of real-time quantitative reverse transcription- PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64 (15): 5245-5250.Search in Google Scholar

ARIOLI, T., L. PENG, A. S. BETZNER, J. BURN, W. WITTKE, W. HERTH, C. CAMILLERI, H. HOFTE, J. PLAZINSKI, R. BIRCH, A. CORK, J. GLOVER, J. REDMOND and R. E. WILLIAMSON (1998): Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279: 717-720.Search in Google Scholar

BALASARAVANAN, T., P. CHEZHIAN, R. KAMALAKANNAN, M. GHOSH, R. YASODHA, M. VARGHESE and K. GURUMURTHI (2005): Determination of inter- and intra-species genetic relationships among six Eucalyptus species based on inter-simple sequence repeats (ISSR). Tree Physiol 25: 1295-1302.Search in Google Scholar

BALASARAVANAN , T., P. CHEZHIAN, R. KAMALAKANNAN, VARGHESE YASODHA, M. VARGHESE, K. GURUMURTHI and M. GHOSH (2006): Identification of species-diagnostic ISSR markers for six Eucalyptus species. Silvae Genet 55(3): 119-122.Search in Google Scholar

BOAVA, L. P., M. L. LAIA, T. R. JACOB, K. M. DABBAS, J. F. GONÇALVES, J. A. FERRO, M. I. T. FERRO and E. L. FUR - TADO (2010): Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using QRT-PCR. BMC Res Notes 3: 43. Search in Google Scholar

BRUNNER, A. M., I. A. YAKOVLEV and S. H. STRAUSS (2004): Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4: 14.Search in Google Scholar

BUSTIN, S. A. (2002): Quantification of mRNA using realtime reverse transcription PCR (RT-PCR) trends and problems. J Mol Endocrinol 29: 23-39.Search in Google Scholar

BUSTIN, S. A. and T. NOLAN (2004): Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. J Biomol Tech 15(3): 155-166.Search in Google Scholar

CHEN, C. Y., M. H. HSIEH, C. C. YANG, C. S LIN and A. WANG (2010): Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii. Phytochemistry 71: 1270-1279.Search in Google Scholar

CHEZHIAN, P., R. YASODHA and M. GHOSH (2010): Genetic diversity analysis in a seed orchard of Eucalyptus tereticornis. New Forest 40(1): 85-99.Search in Google Scholar

CZECHOWSKI, T., M. STITT, T. ALTMAN, M. K. UDVARDI and W. R. SCHEIBLE (2005): Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139(1): 5-17.Search in Google Scholar

DAY, M. E., M. S. GREENWOOD and C. DIAZ-SALA (2002): Age- and size-related trends in woody plant shoot development: regulatory pathways and evidence for genetic control. Tree Physiol 22: 507-513.Search in Google Scholar

DE ALMEIDA, M. R., C. M. RUEDELL, F. K. RICACHENEVSKY, R. A. SPEROTTO, G. PASQUALI and A. G. FETT-NETO (2010): Reference gene selection for quantitative reverse transcription-polymerase chain reaction-normalization during in vitro adventitious rooting in Eucalyptus globulus Labill. BMC Mol Biol 11: 73.Search in Google Scholar

DOBLIN, M. S., I. KURECK, D. JACOB-WILK and D. P. DELMER (2002): Cellulose biosynthesis in plants: from genes to rosettes. Plant Cell Physiol 43: 1407-1420.Search in Google Scholar

EVANS, J. (1992): Plantation forestry in the tropics: In: Tree planting for industrial, social, environmental, and agro forestry purposes, (2nd ed.) Oxford Science Publications, Oxford, New York.Search in Google Scholar

FAGARD, M., T. DESONS, D. F. GOUBET, G. REFREGIER, G. MOUILLE, M. MCCANN, C. RAYON, S. VERNHETTES and H. HOFTE (2000): PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis. Plant Cell 12: 2409-2423.Search in Google Scholar

F.A.O. (2005): Global forest resources assessment 2005 - main report. FAO Forestry Paper, 2005; available online: ftp://ftp.fao.org/docrep/fao/008/A0400E/A0400E00.pdf.Search in Google Scholar

FERNANDEZ, M., C. VILLARROEL, C. BALBONTIN and S. VALENZUELA (2010): Validation of reference genes for real-time QRT-PCR normalization during cold acclimation in Eucalyptus globulus. Trees 24: 1109-1116.Search in Google Scholar

GAN, S., J. SHI, M. LI, K. WU, J. WU and J. BAI (2003): Moderate density molecular maps of Eucalyptus urophylla S.T. Blake and E. tereticornis Smith genomes based on RAPD markers. Genetica 118: 59-67. Search in Google Scholar

GINZINGER, D. G. (2002): Gene quantification using realtime quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30(6): 503-512.Search in Google Scholar

GOICOECHEA, M., E. LACOMBE, S. LEGAY, S. MIHALJEVIC, P. RECH, A. JAUNEAU, C. LAPIERRE, B. POLLET, D. VERHAEGEN, N. C. GIGOT and J. G. PETTENATI (2005): EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis Plant J 43: 553-567.Search in Google Scholar

GUTIERREZ, L., M. MAURIAT, J. PELLOUX, C. BELLINI and O. van WUYTSWINKEL (2008): Towards a systematic validation of references in Real-Time RT-PCR. Plant Cell 20: 1734-1735.Search in Google Scholar

HUGGETT, J., K. DHEDA, S. BUSTIN and A. ZUMLA (2005): Real-time RT-PCR normalization; strategies and considerations. Genes Immun 6(4): 279-284.Search in Google Scholar

IGLESIAS TRABADO, G. and D. WILSTERMANN (2008): Eucalyptus universalis. Global cultivated eucalypt forests map 2008. In: GIT Forestry Consulting’s Eucalyptologics: Information resources on Eucalyptus cultivation worldwide. Retrieved from http://www.git-forestry.comSearch in Google Scholar

ISHITANI, R., K. SUNAGA, A. HIRANO, P. SAUNDERS, N. KATSUBE and D. M. CHUANG (1996): Evidence that glyceraldehyde- 3-phosphate dehydrogenase is involved in ageinduced apoptosis in mature cerebellar neurons in culture. J Neurochem 66(3): 928-935.Search in Google Scholar

JOSHI, C. P. (2003): Molecular biology of cellulose biosynthesis in plants. In: PANDALAI, R. (ed) Recent research developments plant molecular biology. Research Signpost, Kerala, India. p. 19-38.Search in Google Scholar

JOSHI, C. P., S. BHANDARI, P. RANJAN, U. C. KALLURI, X. LIANG, T. FUJINO and A. SAMUGA (2004): Genomics of cellulose biosynthesis in poplars. New Phytol 164: 53-61.Search in Google Scholar

KIMA, H. J., A. BARBARA, TRIPLETTA, H. B. ZHANGB, M. K. LEEB, D. J. C. HINCHLIFFEA, P. LIA and D. D. FANGA (2012): Cloning and characterization of homeologous cellulose synthase catalytic subunit 2 genes from allotetraploid cotton (Gossypium hirsutum L.) Gene 494(2): 181-189.Search in Google Scholar

LEE, P. D., R. SLADEK, C. M. T. GREENWOOD and T. J. HUDSON (2001): Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res 12: 292-297.Search in Google Scholar

LEGAY, S., E. LACOMBE, M. GOICOECHEA, C. BRIERE, A. SEGUIN, J. MACKAY and J. GRIMA-PETTENATI (2007): Molecular characterization of EgMYB1, a putative transcriptional repressor of the lignin biosynthetic pathway. Plant Sci 173: 542-549.Search in Google Scholar

MARQUES, C. M., J. A. ARAUJO, J. G. FERREIRA, R. WHETTEN, D. M. O’MALLEY, H. LIB and R. SEDEROFF (1998): AFLP genetic maps of Eucalyptus globulus and E. tereticornis. Theoret Appl Genet 96: 727-737.Search in Google Scholar

MARUM, L., A. MIGUEL, P. C. RICARDO and C. MIGUEL (2012): Reference Gene Selection for Quantitative Realtime PCR Normalization in Quercus suber. PLoS ONE 7(4): e35113. doi:10.1371/journal.pone.0035113.Search in Google Scholar

MAXWELL, A. (2004): The taxonomy, phylogeny and impact of Mycosphaerella species on eucalypts in south-western Australia. Dissertation, Perth, Murdoch University.Search in Google Scholar

NAIRN, J. and T. HASELKORN (2005): Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms. New Phytol 166: 907-915. Search in Google Scholar

OBEMBE, O. O., E. JACOBSEN, J. P. VINCKEN and R. G. F. VISSER (2009): Differential expression of cellulose synthase (CesA) gene transcripts in potato as revealed by QRT-PCR. Plant Physiol Biochem 47: 1116-1118.Search in Google Scholar

OLBRICH, M., E. GERSTNER, G. WELZL, F. FLEISCHMANN, W. OSSWALD, G. BAHNWEG and D. Z. Ernst (2008): Quantification of mRNAs and housekeeping gene selection for quantitative real-time RT-PCR normalization in European beech (Fagus sylvatica L.) during abiotic and biotic stress. Z. Naturforsch C 63(7-8): 574-82.Search in Google Scholar

PFAFFL, M. W., A. TICHOPAD, C. PRGOMET and T. P. NEUVIANS (2004): Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6): 509-515.Search in Google Scholar

POKE, F. S. (2004): Summary of world-wide research in eucalypt genomics. Prof. R. GRIFFIN, Prof. J. B. REID, Assoc Prof R. Vaillancourt [Consultants Report].Search in Google Scholar

RANIK, M. and A. A. MYBURG (2006): Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cell wall biosynthesis. Tree Physiol 26: 545-556.Search in Google Scholar

REID, K. E., N. OLSSON, J. SCHLOSSER, F. PENG and S. T. LUND (2006): An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6: 27.Search in Google Scholar

SELVEY, S., E.W. THOMPSON, K. MATTHAEI, R. A. LEA, M. G. IRVING and L. R. GRIFFITHS (2001): Beta-actin an unsuitable internal control for RT-PCR. Mol Cell Probes 15(5): 307-311.Search in Google Scholar

SUZUKI, T., P. J. HIGGINS and D. R. CRAWFORD (2000): Control selection for RNA quantitation. Biotechniques 29: 332-337.Search in Google Scholar

TONG, Z., Z. GAO, F. WANG, J. ZHOU and Z. ZHANG (2009): Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10:71 doi:10.1186/1471-2199-10-71.Search in Google Scholar

TURNBULL, J.W. (1999): Eucalypt plantations. New Forest 17: 37-52.Search in Google Scholar

VANDESOMPELE, J., K. DE PRETER, F. PATTYN, B. POPPE, N. VAN ROY, A. de PAEPE and F. SPELEMAN (2002): Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: 1-11.Search in Google Scholar

XU, M., B. ZHANG, S. X. ZHANG and M. HUANG (2011): Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem 408: 337-339.Search in Google Scholar

ZOBEL, B. J., G. V. WYK and P. STAHL (1987): Growing Exotic Forests, pp. 508 in John Wiley and Sons, New York.Search in Google Scholar

Recommended articles from Trend MD

Plan your remote conference with Sciendo