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Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in the Triticeae

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Abstract

Comparative transcriptomics are useful to determine the role of orthologous genes among Triticeae species. Thus they constitute an interesting tool to improve the use of wild relatives for crop breeding. Reverse transcription quantitative real-time PCR (qPCR) is the most accurate measure of gene expression but efficient normalization is required. The choice and optimal number of reference genes must be experimentally determined and the primers optimized for cross-species amplification. Our goal was to test the utility of wheat-reference genes for qPCR normalization when species carrying the following genomes (A, B, D, R, H v and H ch) are compared either simultaneously or in smaller subsets of samples. Wheat/barley/rye consensus primers outperformed wheat-specific ones which indicate that consensus primers should be considered for data normalization in comparative transcriptomics. All genes tested were stable but their ranking in terms of stability differed among subsets of samples. CDC (cell division control protein, AAA-superfamily of ATPases, Ta54227) and RLI (68 kDa protein HP68 similar to Arabidopsis thaliana RNase L inhibitor protein, Ta2776) were always among the three most stable genes. The optimal number of reference genes varied between 2 and 3 depending on the subset of samples and the method used (geNorm vs. coefficient of determination between sequential normalization factors). In any case a maximum number of three reference genes would provide adequate normalization independent of the subset of samples considered. This work constitutes a substantial advance towards comparative transcriptomics using qPCR since it provides useful primers/reference genes.

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Abbreviations

ADP-RF:

ADP-ribosylation factor

CDC :

Cell division control protein, AAA-superfamily of ATPases

NF:

Normalization factor

RLI :

RNase L inhibitor-like protein

qPCR:

Quantitative real-time PCR

Tef-1α:

Translation elongation factor 1α subunit

References

  • Atienza SG, Avila CM, Martin A (2007a) The development of a PCR-based marker for PSY1 from Hordeum chilense, a candidate gene for carotenoid content accumulation in tritordeum seeds. Aust J Agric Res 58:767–773

    Article  CAS  Google Scholar 

  • Atienza SG, Martin AC, Ramirez MC, Martin A, Ballesteros J (2007b) Effects of Hordeum chilense cytoplasm on agronomic traits in common wheat. Plant Breed 126:5–8

    Article  CAS  Google Scholar 

  • Ballesteros J, Ramirez MC, Martinez C, Atienza SG, Martin A (2005) Registration of HT621, a high carotenoid content tritordeum germplasm line. Crop Sci 45:2662–2663

    Article  Google Scholar 

  • Ballesteros J, Cabrera A, Aardse A, Ramirez MC, Atienza SG, Martin A (2007) Registration of TS1, TS10 and TS41, three high biomass production tetraploid triticale germplasm lines. J Plant Reg 1:71–72

    Article  Google Scholar 

  • Barkworth ME, Bothmer Rv (2009) Scientific names in the Triticeae. In: Feuillet C, Muehlbauer GJ (eds) Genetics and genomics of the Triticeae. Springer, Heidelberg, pp 3–30

    Chapter  Google Scholar 

  • Becher M, Talke IN, Krall L, Krämer U (2004) Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant J 37:251–268

    Article  CAS  PubMed  Google Scholar 

  • Bolot S, Abrouk M, Masood-Quraishi U, Stein N, Messing J, Feuillet C, Salse J (2009) The inner circle’ of the cereal genomes. Curr Opin Plant Biol 12:119–125

    Article  CAS  PubMed  Google Scholar 

  • Bru D, Martin-Laurent F, Philippot L (2008) Quantification of the detrimental effect of a single primer-template mismatch by real-time PCR using the 16S rRNA gene as an example. Appl Environ Microbiol 74:1660–1663

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Campoli C, Matus-Cádiz MA, Pozniak CJ, Cattivelli L, Fowler DB (2009) Comparative expression of Cbf genes in the Triticeae under different acclimation induction temperatures. Mol Genet Genomics 282:141–152

    Article  CAS  PubMed  Google Scholar 

  • Chain FJJ, Ilieva D, Evans BJ (2008) Single-species microarrays and comparative transcriptomics. PLoS One 3:e3279

    Article  PubMed  Google Scholar 

  • Cruz F, Kalaoun S, Nobile P, Colombo C, Almeida J, Barros LMG, Romano E, Grossi-de-Sa MF, Vaslin M, Alves-Ferreira M (2009) Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breed 23:607–616

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Feuillet C, Salse J (2009) Comparative genomics in the Triticeae. In: Feuillet C, Muehlbauer GJ (eds) Genetics and genomics of the Triticeae. Springer, Heidelberg, pp 3–30

    Google Scholar 

  • Ghedira R, Papazova N, Vuylsteke M, Ruttink T, Taverniers I, De Loose M (2009) Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification. J Agric Food Chem 57:9370–9377

    Article  CAS  PubMed  Google Scholar 

  • Gil-Humanes J, Piston F, Martin A, Barro F (2009) Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley-wheat amphiploids. BMC Plant Biol 9:66

    Article  PubMed  Google Scholar 

  • Gimenez MJ, Piston F, Martin A, Atienza SG (2010) Application of real-time PCR on the development of molecular markers and to evaluate critical aspects for olive oil authentication. Food Chem 118:482–487

    Article  CAS  Google Scholar 

  • Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487–493

    Article  CAS  PubMed  Google Scholar 

  • Gutierrez L, Mauriat M, Pelloux J, Bellini C, Van Wuytswinkel O (2008) Towards a systematic validation of references in real-time RT-PCR. Plant Cell 20:1734–1735

    Article  CAS  PubMed  Google Scholar 

  • Gutierrez N, Giménez MJ, Palomino C, Avila CM (2010) Assessment of candidate reference genes for expression studies in Vicia faba L. by real-time quantitative PCR. Mol Breed. doi:10.1007/s11032-010-9456-7

  • He P, Friebe BR, Gill BS, Zhou J-M (2003) Allopolyploidy alters gene expression in the highly stable hexaploid wheat. Plant Mol Biol 52:401–414

    Article  CAS  PubMed  Google Scholar 

  • Hibbeler S, Scharsack J, Becker S (2008) Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Mol Biol 9:18

    Article  PubMed  Google Scholar 

  • Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112

    Article  PubMed  Google Scholar 

  • Hu R, Fan C, Li H, Zhang Q, Fu Y-F (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10:93

    Article  PubMed  Google Scholar 

  • Huang XQ, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res 9:868–877

    Article  CAS  PubMed  Google Scholar 

  • Inostroza L, del Pozo A, Matus I, Castillo D, Hayes P, Machado S, Corey A (2009) Association mapping of plant height, yield, and yield stability in recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a Hordeum vulgare subsp. vulgare background. Mol Breed 23:365–376

    Article  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  CAS  PubMed  Google Scholar 

  • Lee J, Roche J, Donaghy D, Thrush A, Sathish P (2010) Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol 11:8

    Article  PubMed  Google Scholar 

  • Maccoux L, Clements D, Salway F, Day P (2007) Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data. BMC Mol Biol 8:62

    Article  PubMed  Google Scholar 

  • Martin AC, Atienza SG, Ramirez MC, Barro F, Martin A (2008) Male fertility restoration of wheat in Hordeum chilense cytoplasm is associated with 6H(ch)S chromosome addition. Aust J Agric Res 59:206–213

    Article  CAS  Google Scholar 

  • Martin AC, Atienza SG, Ramirez MC, Barro F, Martin A (2009) Chromosome engineering in wheat to restore male fertility in the msH1 CMS system. Mol Breed 24:397–408

    Article  CAS  Google Scholar 

  • Martin AC, Atienza SG, Ramirez MC, Barro F, Martin A (2010) Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system. Theor Appl Genet. doi:10.1007/s00122-010-1374-x

  • Matus I, Corey A, Filichkin T, Hayes PM, Vales MI, Kling J, Riera-Lizarazu O, Sato K, Powell W, Waugh R (2003) Development and characterization of recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a Hordeum vulgare subsp. vulgare background. Genome 46:1010–1023

    Article  CAS  PubMed  Google Scholar 

  • Murray YHG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326

    Article  CAS  PubMed  Google Scholar 

  • Paolacci A, Tanzarella O, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:11

    Article  PubMed  Google Scholar 

  • Poole R, Barker G, Wilson I, Coghill J, Edwards K (2007) Measuring global gene expression in polyploidy; a cautionary note from allohexaploid wheat. Funct Integr Genomic 7:207–219

    Article  CAS  Google Scholar 

  • Quackenbush J (2002) Microarray data normalization and transformation. Nat Genet 32:496–501

    Article  CAS  PubMed  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:e45

    Article  CAS  PubMed  Google Scholar 

  • Salentijn E, Goryunova S, Bas N, van der Meer I, van den Broeck H, Bastien T, Gilissen L, Smulders M (2009) Tetraploid and hexaploid wheat varieties reveal large differences in expression of alpha-gliadins from homoeologous Gli-2 loci. BMC Genomics 10:48

    Article  PubMed  Google Scholar 

  • Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063–1066

    Article  CAS  PubMed  Google Scholar 

  • Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M (2010) A practical approach to RT-qPCR–Publishing data that conform to the MIQE guidelines. Methods 50:S1–S5

    Article  CAS  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

    Article  PubMed  Google Scholar 

  • Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J (2006) A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science 314:1298–1301

    Article  CAS  PubMed  Google Scholar 

  • van Ginkel M, Ogbonnaya F (2007) Novel genetic diversity from synthetic wheats in breeding cultivars for changing production conditions. Field Crops Res 104:86–94

    Article  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

    Google Scholar 

  • Villegas D, Casadesús J, Atienza SG, Martos V, Maalouf F, Karam F, Aranjuelo I, Nogués S (2010) Tritordeum, wheat and triticale yield components under multi-local mediterranean drought conditions. Field Crops Res 116:68–74

    Article  Google Scholar 

  • Warburton M, Crossa J, Franco J, Kazi M, Trethowan R, Rajaram S, Pfeiffer W, Zhang P, Dreisigacker S, Ginkel M (2006) Bringing wild relatives back into the family: recovering genetic diversity in CIMMYT improved wheat germplasm. Euphytica 149:289–301

    Article  CAS  Google Scholar 

  • Weber M, Harada E, Vess C, Roepenack-Lahaye Ev, Clemens S (2004) Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J 37:269–281

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was funded by projects (to S.G.A.) AGL2008-03720, P09-AGR-93 from Spanish Ministry of Science (MSI) and Innovation, Junta de Andalucía and FEDER. F. Pistón was the recipient of a Juan de la Cierva Research Grant from MSI.

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Authors and Affiliations

  1. Departamento de Mejora Genética Vegetal, IAS-CSIC, Apdo. 4084, 14080, Córdoba, Spain

    María J. Giménez & Sergio G. Atienza

  2. Departamento de Genética, Universidad de Córdoba, Campus de Rabanales Edif. C-5, 14071, Córdoba, Spain

    Fernando Pistón

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  1. María J. Giménez
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  2. Fernando Pistón
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  3. Sergio G. Atienza
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Correspondence to Sergio G. Atienza.

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Giménez, M.J., Pistón, F. & Atienza, S.G. Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in the Triticeae. Planta 233, 163–173 (2011). https://doi.org/10.1007/s00425-010-1290-y

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