Skip to main content
SpringerLink
Log in

Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (Panicum miliaceum L.)

  • Original Article
  • Published:
Journal of Plant Biology Aims and scope Submit manuscript

Abstract

The appropriate reference genes are crucial for normalization of the target gene expression in qRT-PCR analysis. Broomcorn millet (Panicum miliaceum L.) is one of the most important crops in drought areas worldwide, while the systematical investigation and evaluation of reference genes has not been investigated in this species up to now. Here, 9 commonly used reference genes were selected to detect their expressional stability in different tissues and under different stresses in broomcorn millet. ΔC t , BestKeeper, NormFinder and GeNorm approaches were used to evaluate the potentiality of these candidate genes as the reference gene in broomcorn millet. Taken together, results found that 18S and GAPDH were the suitable reference genes for gene expression normalization in different tissues and under stress treatment in broomcorn millet. This was the first study to investigate the reference genes for qRT-PCR analysis in broomcorn millet, which will facilitate the gene expression studies and also accelerate revealing the molecular mechanism of well-adapted extreme climatic conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Atkinson NJ, Urwin PE (2012) The interaction of plant biotic and abiotic stresses: from genes to the field. J Exp Bot 63:3523–3543

    CAS  PubMed  Google Scholar 

  • Beekman L, Tohver T, Dardari R, Léguillette R (2011) Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol Biol 12:1

    Google Scholar 

  • Bellwood P, Gamble C, Le Blanc SA, Pluciennik M, Richards M, Terrell JE (2007) First Farmers: the Origins of Agricultural Societies. Cambridge Archaeological J 17:87–109

    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

    CAS  PubMed  Google Scholar 

  • Close TJ (1996) Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plantarum 97:795–803

    CAS  Google Scholar 

  • Crawford GW (2006) East Asian plant domestication. Archaeology of Asia:77–95

    Google Scholar 

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

    CAS  Google Scholar 

  • Fuller DQ (2006) Agricultural origins and frontiers in South Asia: a working synthesis. J World Prehistory 20:1–86

    Google Scholar 

  • Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317–325

    CAS  PubMed  Google Scholar 

  • Heller MJ (2002) DNA microarray technology: devices, systems, and applications. Annu Rev Biom Eng 4:129–153

    CAS  Google Scholar 

  • Huang L, Yan H, Jiang X, Yin G, Zhang X, Qi X, Zhang Y, Yan Y, Ma X, Peng Y (2014) Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions. PLoS ONE 9:e93724

    Google Scholar 

  • Hunt HV, Badakshi F, Romanova O, Howe CJ, Jones MK, Heslop-Harrison JP (2014) Reticulate evolution in Panicum (Poaceae): the origin of tetraploid broomcorn millet, P.miliaceum. J Exp Bot 65:3165–3175

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jain M, Chourey PS, Boote KJ, Allen LH (2010) Short-term high temperature growth conditions during vegetative-to-reproductive phase transition irreversibly compromise cell wall invertasemediated sucrose catalysis and microspore meiosis in grain sorghum (Sorghum bicolor). J Plant Physiol 167:578–582

    CAS  PubMed  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 Biop Res Co 345:646–651

    CAS  Google Scholar 

  • Karami A, Shahbazi M, Niknam V, Shobbar ZS, Tafreshi RS, Abedini R, Mabood HE (2013) Expression analysis of dehydrin multigene family across tolerant and susceptible barley (Hordeum vulgare L.) genotypes in response to terminal drought stress. Acta Physiol Plantarum 35:2289–2297

    CAS  Google Scholar 

  • Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biot 17:287–291

    CAS  Google Scholar 

  • Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J App Genet 54:391–406

    CAS  Google Scholar 

  • Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K, Sindelka R, Sjöback R, Sjögreen B, Strömbom L (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125

    CAS  PubMed  Google Scholar 

  • Landi L, Feliziani E, Romanazzi G (2014) Expression of defense genes in strawberry fruits treated with different resistance inducers. J Agr Food Chem 62:3047–3056

    CAS  Google Scholar 

  • Li M-Y, Wang F, Jiang Q, Ma J, Xiong A-S (2014) Identification of SSRs and differentially expressed genes in two cultivars of celery (Apium graveolens L.) by deep transcriptome sequencing. Hort Res 1:10

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Lu H, Zhang J, Liu K-b, Wu N, Li Y, Zhou K, Ye M, Zhang T, Zhang H, Yang X (2009) Earliest domestication of common millet (Panicum miliaceum) in East Asia extended to 10,000 years ago. Proc Nat Acad Sci USA 106:7367–7372

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582

    CAS  PubMed  Google Scholar 

  • Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12:87–98

    CAS  PubMed  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 Biol 10:1

    Google Scholar 

  • Pollier J, Bossche RV, Rischer H, Goossens A (2014) Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus. Plant Physiol Bioch 83:20–25

    CAS  Google Scholar 

  • Ramakers C, Ruijter JM, Deprez RHL, Moorman AF (2003) Assumptionfree analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuros Lett 339:62–66

    CAS  Google Scholar 

  • Ramya M, Reddy KE, Sivakumar M, Pandurangaiah M, Nareshkumar A, Sudhakarbabu O, Veeranagamallaiah G, Sudhakar C (2013) Molecular Cloning, Characterization and Expression Analysis of Stress Responsive Dehydrin Genes from Drought Tolerant Horsegram (Macrotyloma uniflorum (Lam.) Verdc.). Int J Biotech Bioch 9:293–312

    Google Scholar 

  • Rapacz M, Stepien A, Skorupa K (2012) Internal standards for quantitative RT-PCR studies of gene expression under drought treatment in barley (Hordeum vulgare L.): the effects of developmental stage and leaf age. Acta Physiol Plant 34:1723–1733

    CAS  Google Scholar 

  • Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R (2004) When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol 134:1683–1696

    CAS  PubMed  Google Scholar 

  • Schimpl FC, Domingues Júnior AP, de Carvalho Gonçalves JF, da Silva JF, Mazzafera P (2015) References genes for qRT-PCR in guaraná (Paullina cupana var. sorbilis). Brazil J Bot 38:1–8

    Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (1996) Molecular responses to drought and cold stress. Curr Opin Biotech 7:161–167

    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 realtime PCR. BMC Mol Biol 10:71

    PubMed  PubMed Central  Google Scholar 

  • Van den Berg N, Crampton BG, Hein I, Birch PR, Berger DK (2004) High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis. Biotechniques 37:818–24.

    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 Bioch 399:257–261

    CAS  Google Scholar 

  • Wang L, Wang Y, Zhou P (2013) Validation of reference genes for quantitative real-time PCR during Chinese wolfberry fruit development. Plant Physiol Bioch 70:304–310

    CAS  Google Scholar 

  • Wang T, Lu J, Xu Z, Yang W, Wang J, Cheng T, Zhang Q (2014) Selection of suitable reference genes for miRNA expression normalization by qRT-PCR during flower development and different genotypes of Prunus mume. Scientia Horti 169:130–137

    CAS  Google Scholar 

  • Xu Y, Li H, Li X, Lin J, Wang Z, Yang Q, Chang Y (2015) Systematic selection and validation of appropriate reference genes for gene expression studies by quantitative real-time PCR in pear. Acta Physiol Plant 37:1–16

    Google Scholar 

  • Yan J, Yuan F, Long G, Qin L, Deng Z (2012) Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Mol Biol Rep 39:1831–1838

    CAS  PubMed  Google Scholar 

  • Yang H, Liu J, Huang S, Guo T, Deng L, 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

    CAS  PubMed  Google Scholar 

  • Yang Y, He M, Zhu Z, Li S, Xu Y, Zhang C, Singer SD, Wang Y (2012) Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress. BMC Plant Biol 12:1

    Google Scholar 

  • Yeap W-C, Loo JM, Wong YC, Kulaveerasingam H (2014) Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm. Plant Cell, Tiss Org 116:55–66

    CAS  Google Scholar 

  • Zemp N, Minder A, Widmer A (2014) Identification of internal reference genes for gene expression normalization between the two sexes in dioecious white Campion. PLoS ONE 9:e92893

    Google Scholar 

  • Zhang B-c, Sun L, Xiao Z-z, Hu Y-h (2014) Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): internal controls for data normalization. Mar Genom 15:75–84

    Google Scholar 

  • Zhu J, Zhang L, Li W, Han S, Yang W, Qi L (2013) Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions. PLoS ONE 8:e53196

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology in Arid Areas, College of Agronomy and Yangling Branch of China Wheat Improvement Center, Northwest A&F University, Yangling, Shaanxi, 712100, China

    Hong Yue, Pingchuan Deng, Siyan Liu, Meng Wang, Weining Song & Xiaojun Nie

Authors
  1. Hong Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pingchuan Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Siyan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Meng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Weining Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaojun Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Weining Song or Xiaojun Nie.

Additional information

These authors contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yue, H., Deng, P., Liu, S. et al. Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (Panicum miliaceum L.). J. Plant Biol. 59, 435–443 (2016). https://doi.org/10.1007/s12374-016-0024-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12374-016-0024-5

Keywords

Search

Navigation