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Characterizing the expression of translation elongation factor gene EF1α in pear (Pyrus) fruit: evaluation of EF1α as a housekeeping gene

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Abstract

Reference genes are a key factor for the sensitivity and reliability of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The elongation factor gene EF1α encodes a highly conserved ubiquitous protein that functions in the binding of aminoacyl-tRNA to the ribosome during peptide synthesis in eukaryotes, which is proposed for qRT-PCR as an internal reference for its putative housekeeping gene character. However, information about the paralogous copies and expression diversification of EF1α has been neglected and ill defined. In this study, the members of pear (Pyrus) EF1α were explored by mining public data. The family- and/or species-specific members of EF1α in Rosaceae plants or other species revealed by phylogenetic analysis suggested that the specific gene expansion occurred after the family or species diversification. The expression analysis based on the high-throughput sequencing and sequence differences typing provided a high resolution to distinguish the expression among the members of pear EF1α. The EF1α members showed an obviously unstable expression in both pear and apple (Malus × domestica) fruits at different developmental stages, clustered by two conserved expression patterns in pear fruit. The complementary expression among certain pear EF1α members leads to the sum expression of these members having a higher level of expression stability among the fruit at different developmental stages, which can be used as an approach to optimize EF1α as the internal reference. The result can also provide insight into the expression characteristics of pear EF1α in other tissues of pear. In addition, it indicates that any two genes having complementary expression patterns can be used as a reference for qPCR analysis with the high stability of their mean expression value.

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References

  • Bowen J, Ireland HS, Crowhurst R, Luo Z, Watson A, Foster T, Gapper N, Giovanonni J, Mattheis J, Watkins C, Rudell D, Johnston J, Schaffer R (2014) Selection of low-variance expressed Malus × domestica (apple) genes for use as quantitative PCR reference genes (housekeepers). Tree Genet Genomes 10:751–759

    Article  Google Scholar 

  • Chagné D, Crowhurst RN, Pindo M, Thrimawithana A, Deng C, Ireland H, Fiers M, Dzierzon H, Cestaro A, Fontana P, Bianco L, Lu A, Storey R, Knäbel M, Saeed M, Montanari S, Kim YK, Nicolini D, Larger S, Stefani E, Allan AC, Bowen J, Harvey I, Johnston J, Malnoy M, Troggio M, Perchepied L, Sawyer G, Wiedow C, Won K, Viola R, Hellens RP, Brewer L, Bus VG, Schaffer RJ, Gardiner SE, Velasco R (2014) The draft genome sequence of European pear (Pyrus communis L. ‘Bartlett’). PLoS One 9:e92644

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chen J, Li X, Wang D, Li L, Zhou H, Liu Z, Wu J, Wang P, Jiang X, Fabrice MR, Zhang S, Wu J (2015) Identification and testing of reference genes for gene expression analysis in pollen of Pyrus bretschneideri. Scientia Hort 190:43–56

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Evans RC, Campbell CS (2002) The origin of the apple subfamily (Maloideae; Rosaceae) is clarified by DNA sequence data from duplicated GBSSI genes. Amer J Bot 89:1478–1484

    Article  CAS  Google Scholar 

  • Fuentes A, Ortiz J, Saavedra N, Salazar LA, Meneses C, Arriagada C (2016) Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis. Plant Physiol Biochem 101:124–131

    Article  PubMed  CAS  Google Scholar 

  • Granados JM, Ávila C, Cánovas FM, Cañas RA (2016) Selection and testing of reference genes for accurate RT-qPCR in adult needles and seedlings of maritime pine. Tree Genet Genomes 12:60

    Article  Google Scholar 

  • Guénin 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  PubMed  CAS  Google Scholar 

  • Gutierrez L, Mauriat M, Guénin 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 6:609–618

    Article  PubMed  CAS  Google Scholar 

  • Huang X, Li K, Xu X, Yao Z, Jin C, Zhang S (2015) Genome-wide analysis of WRKY transcription factors in white pear (Pyrus bretschneideri) reveals evolution and patterns under drought stress. BMC Genomics 16:1104

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Imai T, Ubi BE, Saito T, Moriguchi T (2014) Evaluation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in Pyrus pyrifolia using different tissue samples and seasonal conditions. PLoS One 9:e86492

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Janssen BJ, Thodey K, Schaffer RJ, Alba R, Balakrishnan L, Bishop R, Bowen JH, Crowhurst RN, Gleave AP, Ledger S, McArtney S, Pichler FB, Snowden KC, Ward S (2008) Global gene expression analysis of apple fruit development from the floral bud to ripe fruit. BMC Plant Biol 8:16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Jung S, Cestaro A, Troggio M, Main D, Zheng P, Cho I, Folta KM, Sosinski B, Abbott A, Celton JM, Arús P, Shulaev V, Verde I, Morgante M, Rokhsar D, Velasco R, Sargent DJ (2012) Whole genome comparisons of Fragaria, Prunus and Malus reveal different modes of evolution between rosaceous subfamilies. BMC Genomics 13:129

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lin Y, Zhang C, Lan H, Gao S, Liu H, Liu J, Cao M, Pan G, Rong T, Zhang S (2014) Validation of potential reference genes for qPCR in maize across abiotic stresses, hormone treatments, and tissue types. PLoS One 9:e95445

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Malnoy M, Faize M, Venisse JS, Geider K, Chevreau E (2005) Expression of viral EPS-depolymerase reduces fire blight susceptibility in transgenic pear. Plant Cell Rep 23:632–638

    Article  PubMed  CAS  Google Scholar 

  • Morga B, Arzul I, Faury N, Renault T (2010) Identification of genes from flat oyster Ostrea edulis as suitable housekeeping genes for quantitative real time PCR. Fish Shellfish Immunol 29:937–945

    Article  PubMed  CAS  Google Scholar 

  • Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1:1–2

    Article  PubMed  PubMed Central  Google Scholar 

  • Negrutskii BS, El’skaya AV (1998) Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucl Acid Res Mol Biol 60:47–78

    Article  CAS  Google Scholar 

  • Niu Q, Li J, Cai D, Qian M, Jia H, Bai S, Hussain S, Liu G, Teng Y, Zheng X (2016) Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud. J Exp Bot 67:239–257

    Article  PubMed  CAS  Google Scholar 

  • Roger AJ, Sandblom O, Doolittle WF, Philippe H (1999) An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes. Mol Biol Evol 16:218–233

    Article  PubMed  CAS  Google Scholar 

  • Saraiva KD, Fernandes de Melo D, Morais VD, Vasconcelos IM, Costa JH (2014) Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions. Plant Cell Rep 33:1453–1465

    Article  PubMed  CAS  Google Scholar 

  • Schaffer RJ, Friel EN, Souleyre EJ, Bolitho K, Thodey K, Ledger S, Bowen JH, Ma JH, Nain B, Cohen D, Gleave AP, Crowhurst RN, Janssen BJ, Yao JL, Newcomb RD (2007) A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway. Plant Physiol 144:1899–1912

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Shivhare R, Lata C (2016) Selection of suitable reference genes for assessing gene expression in pearl millet under different abiotic stresses and their combinations. Sci Rep 6:23036

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald LM, Gutin N, Lanchbury J, Macalma T, Mitchell JT, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu VT, King ST, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater MM, Masiero S, Lasserre P, Lespinasse Y, Allan AC, Bus V, Chagn é D, Crowhurst RN, Gleave AP, Lavezzo E, Fawcett JA, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens RP, Durel CE, Gutin A, Bumgarner RE, Gardiner SE, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R (2010) The genome of the domesticated apple (Malus ×domestica Borkh.). Nature Genet 42:833–839

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Dai M, Zhang S, Shi Z (2013) Exploring the hormonal and molecular regulation of sand pear (Pyrus pyrifolia) seed dormancy. Seed Sci Res 23:15–25

    Article  CAS  Google Scholar 

  • Wang Y, Dai M, Zhang S, Shi Z (2014) Exploring candidate genes for pericarp russet pigmentation of sand pear (Pyrus pyrifolia) via RNA-Seq data in two genotypes contrasting for pericarp color. PLoS One 9:e83675

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Rev Genet 10:57–63

    Article  PubMed  CAS  Google Scholar 

  • Watillon B, Kettmann R, Boxus P, Burny A (1998) Elongation factor 1α (EF1α) transcript levels are developmentally and environmentally regulated in apple plants. Physiol Plantarum 104:1–9

    Article  CAS  Google Scholar 

  • Wu J, Li LT, Li M, Khan MA, Li XG, Chen H, Yin H, Zhang SL (2014) High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers. J Exp Bot 65:5771–5781

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wu J, Wang Z, Shi Z, Zhang S, Ming R, Zhu S, Khan MA, Tao S, Korban SS, Wang H, Chen NJ, Nishio T, Xu X, Cong L, Qi K, Huang X, Wang Y, Zhao X, Wu J, Deng C, Gou C, Zhou W, Yin H, Qin G, Sha Y, Tao Y, Chen H, Yang Y, Song Y, Zhan D, Wang J, Li L, Dai M, Gu C, Wang Y, Shi D, Wang X, Zhang H, Zeng L, Zheng D, Wang C, Chen M, Wang G, Xie L, Sovero V, Sha S, Huang W, Zhang S, Zhang M, Sun J, Xu L, Li Y, Liu X, Li Q, Shen J, Wang J, Paull RE, Bennetzen JL, Wang J, Zhang S (2013) The genome of the pear (Pyrus bretschneideri Rehd.). Genome Res 23:396–408

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wu T, Zhang R, Gu C, Wu J, Wan H, Zhang S, Zhang S (2012) Evaluation of candidate reference genes for real time quantitative PCR normalization in pear fruit. African J Agr Res 7:3701–3704

    Google Scholar 

  • Xiao X, Ma J, Wang J, Wu X, Li P, Yao Y (2015) Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR. Front Plant Sci 5:788

    Article  PubMed  PubMed Central  Google Scholar 

  • Xu Y, Li H, Li X, Lin J, Wang Z, Yang Q, Chang Y (2015) Systematic selection and validation of ppropriate reference genes for gene expression studies by quantitative real-time PCR in pear. Acta Physiol Plant 37:40

    Article  CAS  Google Scholar 

  • Zhang J (2003) Evolution by gene duplication: an update. Trends Ecol Evol 18:292–298

    Article  Google Scholar 

  • Zhang S, Wu J, Chen H, Gu C, Tao S, Wu J, Zhang S (2011) Identification of differentially expressed genes in a spontaneous mutant of ‘Nanguoli’ pear (Pyrus ussuriensis Maxim) with large fruit. J Hort Sci Biotech 86:595–602

    Article  CAS  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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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  1. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, Zhejiang Province, China

    Yue-zhi Wang, Mei-song Dai, Dan-ying Cai, Lixiang Miao, Lingzhu Wei & Ze-bin Shi

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  1. Yue-zhi Wang
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  2. Mei-song Dai
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  3. Dan-ying Cai
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  4. Lixiang Miao
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Correspondence to Ze-bin Shi.

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Communicated by D. Chagné

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Supplementary Fig. S1

Expression of pear translation elongation factor genes (EF1α) estimated by fragments per kilobase of exon per million fragments mapped (FPKM) based on the high-throughput sequencing analysis. The transcript abundance for each gene was gene symbols PpEF1α-1 to PpEF1α-7 correspond to Pbr030904.1, Pbr030912.1, Pbr029028.1, Pbr034452.1, Pbr034447.1, Pbr029031.1, and Pbr033900.1, respectively. For each gene, the arithmetic means were calculated for the biological replicates. The expression data were presented as mean ± standard deviation. The P values for expression data were calculated between 0 d After Anthesis (DAA) (type 1) and 14, 30, 60, and 100 DAA (types 2–5) using the Student t test (◊, P < 0.05; ♦, P < 0.01). (PNG 373 kb)

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Wang, Yz., Dai, Ms., Cai, Dy. et al. Characterizing the expression of translation elongation factor gene EF1α in pear (Pyrus) fruit: evaluation of EF1α as a housekeeping gene. Tree Genetics & Genomes 14, 62 (2018). https://doi.org/10.1007/s11295-018-1268-7

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  • DOI: https://doi.org/10.1007/s11295-018-1268-7

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