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Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

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Abstract

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C q) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

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References

  • Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274. doi:10.1007/s00299-006-0204-8

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2

    PubMed  CAS  Google Scholar 

  • 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. doi:10.1158/0008-5472.CAN-04-0496

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193. doi:10.1677/jme.0.0250169

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39. doi:10.1677/jme.0.0290023

    Article  PubMed  CAS  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer C (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. doi:10.1373/clinchem.2008.112797

    Article  PubMed  CAS  Google Scholar 

  • Chen M, Wang QY, Cheng XG, Xu ZS, Li LC, Ye XG, Xia LQ, Ma YZ (2007) GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-salt tolerance in transgenic plants. Biochem Biophys Res Commun 353:299–305. doi:10.1016/j.bbrc.2006.12.02

    Article  PubMed  CAS  Google Scholar 

  • CIAT (International Center for Tropical Agriculture) (1990) Research constraints provisionally identified by CIAT. In: Workshop on advanced Phaseolus bean research network, Cali, p 30

  • Cordoba EM, Die JV, González-Verdejo CI, Nadal S, Román B (2011) Selection of reference genes in Hedysarum coronarium under various stresses and stages of development. Anal Biochem 409:236–243. doi:10.1016/j.ab.2010.10.031

    Article  PubMed  CAS  Google Scholar 

  • Cunningham DF, O’Connor B (1997) Proline-specific peptidases. Biochim Biophys Acta 1343:160–186. doi:10.1016/S0167-4838(97)00134-9

    Article  PubMed  CAS  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17. doi:10.1104/pp.105.063743

    Article  PubMed  CAS  Google Scholar 

  • Expósito-Rodríguez M, Borges AA, Borges-Perez A, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131. doi:10.1186/1471-2229-8-131

    Article  PubMed  Google Scholar 

  • Ferreira RB, Monteiro S, Freitas R, Santos CN, Chen Z, Batista LM, Duarte J, Borges A, Teixeira AR (2007) The role of plant defence proteins in fungal pathogenesis. Mol Plant Pathol 8:677–700. doi:10.1111/j.1364-3703.2007.00419.x

    Article  PubMed  CAS  Google Scholar 

  • Gepts P, Aragao F, Barros E, Blair MW, Brondani R, Broughton W, Galasso I, Hernández G, Kami J, Lariguet P, McClean P, Melotto M, Miklas P, Pauls P, Pedrosa-Harand A, Porch T, Sánchez F, Sparvoli F, Yu K (2008) Genomics of Phaseolus beans, a major source of dietary protein and micronutrients in the tropics. In: Moore PH, Ming R (eds) Genomics of tropical crops. Springer, New York, pp 113–143

    Chapter  Google Scholar 

  • Guimarães CM (1992) Características morfo-fisiológicas do feijoeiro (Phaseolus vulgaris L.) relacionadas com a resistência à seca. PhD Thesis, Unicamp, Brazil

  • Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6(6):609–618. doi:10.1111/j.1467-7652.2008.00346

    Article  PubMed  CAS  Google Scholar 

  • Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994. doi:10.1101/gr.6.10.986

    Article  PubMed  CAS  Google Scholar 

  • Hu R, Fan C, Li H, Zhang Q, Fu YF (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Bio 10:93. doi:10.1186/1471-2199-10-93

    Article  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 345:646–651. doi:10.1016/j.bbrc.2006.04.140

    Article  PubMed  CAS  Google Scholar 

  • Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59. doi:10.1186/1471-2199-9-59

    Article  PubMed  Google Scholar 

  • Karlen Y, Mcnair A, Perseguers S, Mazza C, Mermod N (2007) Statistical significance of quantitative PCR. BMC Bioinforma 8:131. doi:10.1186/1471-2105-8-131

    Article  Google Scholar 

  • Kulcheski FR, Marcelino-Guimaraes FC, Nepomuceno AL, Abdelnoor RV, Margis R (2010) The use of microRNA as reference genes for quantitative polymerase chain reaction in soybean. Anal Biochem 406:185–192. doi:10.1016/j.ab.2010.07.020

    Article  PubMed  CAS  Google Scholar 

  • Libault M, Thibivilliers S, Bilgin DD, Radwan O, Benitez M, Clough SJ, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54. doi:10.3835/plantgenome2008.02.0091

    Article  CAS  Google Scholar 

  • Lovdal T, Lillo C (2009) Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387:238–242. doi:10.1016/j.ab.2009.01.024

    Article  PubMed  CAS  Google Scholar 

  • Maroufi A, Bockstaele EV, Loose MD (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:1. doi:10.1186/1471-2199-11-15

    Article  Google Scholar 

  • Melotto M, Kelly JD (2000) An allelic series at the Co-1 locus conditioning resistance to anthracnose in common bean of Andean origin. Euphyt 116:143–149. doi:10.1023/A:1004005001049

    Article  Google Scholar 

  • Melotto M, Monteiro-Vitorello CB, Bruschi AG, Camargo LE (2005) Comparative bioinformatic analysis of genes expressed in common bean (Phaseolus vulgaris L.) seedlings. Genome 48:562–570. doi:10.1139/G05-010

    Article  PubMed  CAS  Google Scholar 

  • Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Bio 10:11. doi:10.1186/1471-2199-10-11

    Article  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45. doi:10.1093/nar/29.9.e45

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acid Res 30(9):e36. doi:10.1093/nar/30.9.e36

    Article  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 339:62–66. doi:10.1016/S0304-3940(02)01423-4

    Article  PubMed  CAS  Google Scholar 

  • Reece RJ (2004) Analysis of genes and genomes. Wiley, Chichester

    Google Scholar 

  • Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, Van Den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37(6):e45. doi:10.1093/nar/gkp045

    Article  PubMed  CAS  Google Scholar 

  • Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291–295. doi:10.1016/S0168-1656(99)00163-7

    Article  PubMed  CAS  Google Scholar 

  • Thibivilliers S, Joshi T, Campbell K, Scheffler B, Xu D, Cooper B, Nguyen H, Stacey G (2009) Generation of Phaseolus vulgaris ESTs and investigation of their regulation upon Uromyces appendiculatus infection. BMC Plant Biol 9:46. doi:10.1186/1471-2229-9-46

    Article  PubMed  Google Scholar 

  • Tong Z, Gao Z, Wang F, Zhou J, Zhang Z (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

    Article  PubMed  Google Scholar 

  • Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C (2002) Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 309:293–300. doi:10.1016/S0003-2697(02)00311-1

    Article  PubMed  CAS  Google Scholar 

  • Tunbridge EM, Eastwood SL, Harrison PJ (2011) Changed relative to what? housekeeping genes and normalization strategies in human brain gene expression studies. Biol Psychiatry 69:173–179. doi:10.1016/j.biopsych.2010.05.023

    Article  PubMed  CAS  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:research0034-research0034.11. doi:10.1186/gb-2002-3-7-research0034

  • Zhong H-Y, Chen J-W, Li C-Q, Chen L, Wu J-Y, Chen J-Y, Lu W-J, Li J-G (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 30:641–653. doi:10.1007/s00299-010-0992-8

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We greatly thank Dr. Adriane Wendland from the National Rice and Beans Research Center (EMBRAPA), Goiás, Brazil, for providing strains of the fungus; the Center for Phytosanitary Research and Development (IAC), Campinas, Brazil, for the preparation of fungus inoculum; the Center for Analysis and Technological Research of Grain and Fiber Agribusiness (IAC), Campinas, Brazil, for verifying the pathogen race; CNPq (National Council for Scientific and Technological Development) for the scholarship and financial support (Universal-474337/2008-1); and CAPES for the post-doctoral fellowship.

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The authors declare that they have no conflicts of interest.

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  1. Center of Nuclear Energy for Agriculture, Laboratory of Cellular and Molecular Biology, University of São Paulo, P. O. Box 96 CEP, Piracicaba, SP, 13400-970, Brazil

    Aline Borges, Siu Mui Tsai & Danielle Gregorio Gomes Caldas

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  1. Aline Borges
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  2. Siu Mui Tsai
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  3. Danielle Gregorio Gomes Caldas
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Correspondence to Danielle Gregorio Gomes Caldas.

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Communicated by Q. Zhao.

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Borges, A., Tsai, S.M. & Caldas, D.G.G. Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses. Plant Cell Rep 31, 827–838 (2012). https://doi.org/10.1007/s00299-011-1204-x

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  • DOI: https://doi.org/10.1007/s00299-011-1204-x

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