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Reference genes selection for quantitative gene expression studies in Pinus massoniana L.

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Abstract

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Evaluation and selection of reference genes in Pinus massoniana L. (PM) for gene expression studies of various tissues, floral organ development, and abiotic stress.

Abstract

An important prerequisite for obtaining accurate gene expression results using quantitative real-time PCR is the selection of a reference gene or a group of genes having a highly stable level of expression. Pinus massoniana L. (PM) is the predominant fast-growing timber forest tree species in southern China. In this study of PM, we evaluated various tissues, flowers in different developmental phases, leaves from a cultivar with insect resistance, and leaves from plants under several types of abiotic stresses. Comprehensive Analysis was performed using BestKeeper, Normfinder, geNorm, and RefFinder software to select the most stable reference gene or gene group from among 25 candidate genes in these samples. The results showed that different experimental conditions require the use of different reference genes: ACT1 could be used as a reference gene for all samples in this study; UBI4 was the best gene for various tissues and zinc stress; CYP was the most stable gene for leaves from insect-resistant materials and Pb stress; Fbox and UBI11 were the best reference genes for salt stress; Fbox + RRP8, ARF + TUBA, and EF1B + IDH were the best reference groups for drought stress, low temperature stress, and flowers in different developmental phases, respectively. This study presents a reliable selection of reference genes for Masson pine, and the conclusions are meaningful for improving the accuracy of expression analyses in future molecular biology studies.

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References

  • Andersen CL, Jensen JL, Orntoft TF (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 64:5245–5250

    Article  CAS  PubMed  Google Scholar 

  • Artico S, Nardeli SM, Brilhante O, Grossi-de-Sa MF, Alves-Ferreira M (2010) Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol 10:49

    Article  PubMed  PubMed Central  Google Scholar 

  • Boava LP, Laia ML, Jacob TR, Dabbas KM, Gonc alves JF, Ferro JA, Ferro MI, Furtado EL (2010) Selection of endogenous genes for gene expression studiesin Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR. BMC Res Notes 3:43

  • Brunner AM, Yakovlev IA, Strauss SH (2004) Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 18(4):14

    Article  Google Scholar 

  • Cassan-Wang H, Soler M, Yu H, Camargo EL, Carocha V, Ladouce N, Savelli B, Paiva JA, Leplé JC, Grima-Pettenati J (2012) Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions. Plant Cell Physiol 53(12):2101–2116

    Article  CAS  PubMed  Google Scholar 

  • Chang E, Shi S, Liu J, Cheng T, Xue L, Yang X, Yang W, Lan Q, Jiang Z (2012) Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR. PLoS One 7(3):e33278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen D, Pan X, Xiao P, Farwell MA, Zhang B (2011a) Evaluation and identification of reliable reference genes for pharmogenomics, toxicogenomics and small RNA expression analysis. J Cell Physiol 226:2469–2477

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Zhong HY, Kuang JF, Li JG, Lu WJ, Chen JY (2011b) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234(2):377–390

    Article  CAS  PubMed  Google Scholar 

  • Chen H, Yang ZQ, Tan JH, Feng YH, Jia J, Tang GQ, Ou J (2015) Molecular cloning PmFT1 gene and its effects on floral development of Pinus massoniana. Genom Appl Biol 34(4):806–812

    CAS  Google Scholar 

  • Chou IT, Gasser CS (1997) Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of know ncyclophilin proteins. Plant Mol Biol 35:873–892

    Article  CAS  PubMed  Google Scholar 

  • Cornejo I, Sepulveda FV, Kibenge FSB, Young JI (2010) Isolation of the Atlantic salmon β-actin promoter and its use to drive expression in salmon cells in culture and in transgenic zebra fish. Aquaculture 309(1–4):75–81

    Article  CAS  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genomewide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139(1):5–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • de Almeida MR, Ruedell CM, Ricachenevsky FK, Sperotto RA, Pasquali G, Fett-Neto AG (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

    Article  PubMed  PubMed Central  Google Scholar 

  • de Oliveira LA, Breton MC, Bastolla FM, Camargo Sda S, Margis R, Frazzon J, Pasquali G (2012) Reference genes for the normalization of gene expression in eucalyptus species. Plant Cell Physiol 53(2):405–422

    Article  PubMed  Google Scholar 

  • Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344(1):141–143

    Article  CAS  PubMed  Google Scholar 

  • Die JV, Román B, Nadal S, González-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232:145–153

    Article  CAS  PubMed  Google Scholar 

  • Dubrovina AS, Kiselev KV, Khristenko VS (2013) Expression of calciumdependent protein kinase (CDPK) genes under abiotic stress conditions in wildgrowing grapevine Vitis amurensis. J Plant Physiol 170:1491–1500

    Article  CAS  PubMed  Google Scholar 

  • Exposito-Rodriguez M, Borges A, Borges-Perez A, Perez J (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131

    Article  PubMed  PubMed Central  Google Scholar 

  • Fernandes J, Morrow DJ, Casati P, Walbot V (2008) Distinctive transcriptome responses to adverse environmental conditions in Zea mays L. Plant Biotechnol J 6(8):782–798

    Article  CAS  PubMed  Google Scholar 

  • Gao Y, Xi ML, Wang GF, Yang LW, Shi JS (2010) Molecular characterization and expression analysis of PmSERK1 during somatic embryogenesis in Masson Pine. Mol Plant Breed 8(1):53–58 (in chinese)

    Google Scholar 

  • Gubern C, Hurtado O, Rodríguez R, Morales JR, Romera VG, Moro MA, Liza-soain I, Serena J, Mallolas J (2009) Validation of housekeeping genes for quantitative real-time PCR in in vivo and in vitro models of cerebral ischaemia. BMC Mol Biol 10:57–67

    Article  PubMed  PubMed Central  Google Scholar 

  • Gutierrez L, Mauriat M, Gunin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Wuytswinkel OV (2008) The lack of a systematic validation of reference genes: a serious pitfall under-valued in reverse transcription polymerase chain reaction (RT PCR) analysis in plants. Plant Biotechno1 J 6(6):609–618

  • Han WP, Ding GJ, Bao B (2012) Physiological and ecological responses of Pinus massoniana seedling from different provenances to drought stress. J Cent South Univ For Technol 32(5):25–29 (In Chinese)

    CAS  Google Scholar 

  • Hu Y, He XH, Chen H, Luo C, Dong L, Zhang SW, Huang GX (2014) Election of reference genes for real-time quantitative PCR studies of kumquat in various tissues and under abiotic stress. Sci Hortic 174:207–216

    Article  CAS  Google Scholar 

  • Ikeda F, Dikic I (2008) Atypical ubiquitin chains: new molecular signals. ‘Protein Modifi cations: Beyond the Usual Suspects’ review series. EMB O Rep 9(6):536–542

  • Iskandar HM, Simpson RS, Casu RE, Bonnett GD, Maclean DJ, Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Rep 22:325–337

    Article  CAS  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345(2):646–651

    Article  CAS  PubMed  Google Scholar 

  • Jian B, Liu B, Bi YR, Hou WS, Wu CX, Han TF (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9(1):59–63

    Article  PubMed  PubMed Central  Google Scholar 

  • Kumar V, Sharma R, Trivedi PC, Vyas GK, Khandel W (2011) Traditional and novel references towards systematic normalization of qRT-PCR data in plants. Aust J Crop Sci 5(11):1455–1468

    Google Scholar 

  • Lee SA, Yoon EK, Heo JO, Lee MH, Hwang I, Cheong H, Lee WS, Hwang YS, Lim J (2012) Analysis of Arabidopsis glucose insensitive growth mutants reveals the involvement of the plastidial copper transporter PAA1 in glucose-induced intracellular signaling. Plant Physiol 159(3):1001–1012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li W, Qian YQ, Han L, Liu JX, Sun ZY (2014) Identification of suitable reference genes in buffalo grass for accurate transcript normalization under various abiotic stress conditions. Gene 547:55–62

    Article  CAS  PubMed  Google Scholar 

  • Lilly ST, Drummond RSM, Pearson MN, Macdiarmid RM (2011) Identification and validation of reference genes for normalization of transcripts from Virus-Infected arabidopsis thaliana. Am Phytopathol Soc 24(3):294–304

    CAS  Google Scholar 

  • Lin YL, Lai ZX (2010) Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Sci 178(4):359–365

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Long XY, Wang JR, Ouellet T, Rocheleau H, Wei YM, Pu ZE, Jiang QT, Lan XJ, Zheng YL (2010) Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat. Plant Mol Biol 74:307–311

    Article  CAS  PubMed  Google Scholar 

  • Mafra V, Kubo KS, Alves-Ferreira M, Ribeiro-Alves M, Stuart RM, Boava LP, Rodrigues CM, Machado MA (2012) Reference genes for accurate transcript normalizationin citrus genotypes under different experimental conditions. PLoS One 7(2):e31263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marum L, Miguel A, Ricardo CP, Miguel C (2012) Reference gene selection for quantitative Real-time PCR normalization in quercus suber. PLoS One 7(4):e35113. doi:10.1371/journal.pone.0035113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mascia T, Santovito E, Gallitelli D, Cillo F (2010) Evaluation of reference genes for quantitative reverse transcription polymerase chain reaction normalization in infected tomato plants. Mol Plant Pathol 11:805–816

    Article  CAS  PubMed  Google Scholar 

  • Moura JC, Araújo P, Brito Mdos S, Souza UR, Viana Jde O, Mazzafera P (2012) Validation of reference genes from Eucalyptus spp. under different stress conditions. BMC Res Notes 14(5):634

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Article  CAS  PubMed  Google Scholar 

  • Pettengill EA, Parmentier-Line C, Coleman GD (2012) Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies. BMC Res Notes. 23(5):366

    Article  Google Scholar 

  • Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515

    Article  CAS  PubMed  Google Scholar 

  • Phillips MA, D’Auria JC, Luck K, Gershenzon J (2009) Evaluation of Candidate reference genes for real-time quantitative PCR of plant samples using purified cDNA as template. Plant Mol Biol Rep 27(3):407–416

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Popovici V, Goldstein DR, Antonov J, Jaggi R, Delorenzi M, Wirapati P (2009) Selecting control genes for RT-QPCR using public microarray data. BMC Bioinform 10(1):42–52

    Article  Google Scholar 

  • Radonic A, Thulke S, Mackay IM, Landt O, Siegert W et al (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862

    Article  CAS  PubMed  Google Scholar 

  • Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339(1):62–66

    Article  CAS  PubMed  Google Scholar 

  • Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (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

    Article  PubMed  PubMed Central  Google Scholar 

  • Schmidt G, Delaney S (2010) Stable internal reference genes for normalization of Real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283(3):233–241

    Article  CAS  PubMed  Google Scholar 

  • Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46(1–2):69–81

    Article  CAS  PubMed  Google Scholar 

  • Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33

    Article  PubMed  PubMed Central  Google Scholar 

  • Srivastava S, Chen L (2010) A two-parameter generalized poisson model to improve the analysis of RNA-seq data. Nucl Acids Res 38(15):e170

    Article  PubMed  PubMed Central  Google Scholar 

  • Stürzenbaum SR, Kille P (2001) Control genes in quantitative molecular biological techniques: the variability of invariance. Comp Biochem Physiol B 130(3):281–289

    Article  PubMed  Google Scholar 

  • Tan JH (2013) Physiological and biochemical responses of Pinus massoniana superior provenance seedlings under artificial low temperature stress. Scientia Silvae Sincae 49(3):51–55 (In Chinese)

    CAS  Google Scholar 

  • Tong ZG, Gao ZH, Wang F, Zhou HJ, Zhang Z (2009) Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol 10(1):71–83

    Article  PubMed  PubMed Central  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7):Research00-12

  • VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantita-tive PCR for gene expression analysis. Biotechniques 44(5):619–626

    Article  CAS  PubMed  Google Scholar 

  • Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399(2):257–261

    Article  CAS  PubMed  Google Scholar 

  • Wang M, Wang QL, Zhang BH (2013) Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.). Gene 530:44–50

    Article  CAS  PubMed  Google Scholar 

  • Wang HL, Chen J, Tian Q, Wang S, Xia X, Yin W (2014) Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR. Physiol Plant 152(3):529–545

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Yu K, Poysa V, Shi C, Zhou Y (2012) Selection of reference genes for normalization of qRT-PCR analysis of differentially expressed genes in soybean exposed to cadmium. Mol Biol Rep 39(2):1585–1594

    Article  CAS  PubMed  Google Scholar 

  • Wu RJ, Zhuang J, Huang J, Chen WP (2009) Responses and resistance mechanism of Pinus massoniana under the stresses of simulated acid rain and aluminum. Scientia Silvae Sincae 45(12):22–29 (In Chinese)

    CAS  Google Scholar 

  • Xie F, Sun G, Stiller JW, Zhang B (2011) Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database. PLoS One 6(11):e26980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu M, Zhang B, Su X, Zhang S, Huang M (2011) Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem 408(2):337–339

    Article  CAS  PubMed  Google Scholar 

  • Xu L, Li ZY, Lu Q, Liang J, Zhang XY (2014) Cloning and expression characteristics of Pinus massoniana pmCaM response to bursaphelenchus xylophilus infection. Forest Research 27(3):323–328 (in chinese)

    Google Scholar 

  • Yang CG, Wang XL, Tian J, Liu W, Wu F, Jiang M, Wen H (2013) Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus). Gene 527:83–192

    Google Scholar 

  • Yang HL, Liu J, Huang SM, Guo TT, Deng LB, Hua W (2014) Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L. Gene 538:113–122

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, He LL, Fu QT, Xu ZF (2013) Selection of reliable reference genes for gene expression studies in the biofuel plant Jatropha curca s using real-time quantitative PCR. Int J Mol Sci 14:24338–24354

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhong HY, Chen JW, Li CQ, Chen L, Wu JY, Chen JY, Lu WJ, Li JG (2011) Selection of reliable reference genes for expression studies by reverse transcription quantitative Real- time PCR in litchi under different experimental conditions. Plant Cell Rep 30(4):641–653

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This project is partially supported by The special fund for Bagui scholar (2011A015) and Natural Science Foundation of Guangxi Zhuang Autonomous Region (Grant No. 2014jjBA30102) and Basic scientific research fund of Guangxi Forestry Research Institute-China (Grant No. Linke 201410) and Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation (13-A-03-02; 13B0301) and Key Scientific and Technological project of Guangxi Zhuang Autonomous Region (Grant No. 1123004-4A).

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Authors and Affiliations

  1. Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Guangxi Forestry Research Institute of Science, Nanning, 530002, People’s Republic of China

    Hu Chen, Zhangqi Yang, Jianhui Tan, Jie Jia & Hulan Xu

  2. Guangxi University, Nanning, 530004, People’s Republic of China

    Ying Hu & Xinhua Chen

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  1. Hu Chen
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  2. Zhangqi Yang
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  3. Ying Hu
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  4. Jianhui Tan
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  7. Xinhua Chen
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Correspondence to Zhangqi Yang.

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Chen, H., Yang, Z., Hu, Y. et al. Reference genes selection for quantitative gene expression studies in Pinus massoniana L.. Trees 30, 685–696 (2016). https://doi.org/10.1007/s00468-015-1311-3

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