Selection of optimal reference genes for qRT-PCR analysis of shoot development and graviresponse in prostrate and erect chrysanthemums


Autoři: Xiaowei Li aff001;  Yujie Yang aff001;  Sagheer Ahmad aff001;  Ming Sun aff001;  Cunquan Yuan aff001;  Tangchun Zheng aff001;  Yu Han aff001;  Tangren Cheng aff001;  Jia Wang aff001;  Qixiang Zhang aff001
Působiště autorů: Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural E aff001;  Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, Beijing Laboratory of Urban and Rural E aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225241

Souhrn

The prostrate cultivars of ground-cover chrysanthemum have been used in landscape gardening due to their small stature, large crown width and strong branching ability. qRT-PCR is a rapid and powerful tool for gene expression analysis, while its accuracy highly depends on the stability of reference genes. The paucity of authentic reference genes presents a major hurdle in understanding the genetic regulators of prostrate architecture. Therefore, in order to reveal the regulatory mechanism of prostrate growth of chrysanthemum stems, here, stable reference genes were selected for expression analysis of key genes involved in shoot development and graviresponse. Based on transcriptome data, eleven reference genes with relatively stable expression were identified as the candidate reference genes. After the comprehensive analysis of the stability of these reference genes with four programs (geNorm, NormFinder, BestKeeper and RefFinder), we found that TIP41 was the most stable reference gene in all of the samples. SAND was determined as a superior reference gene in different genotypes and during the process of shoot development. The optimal reference gene for gravitropic response was PP2A-1. In addition, the expression patterns of LA1 and PIN1 further verified the reliability of the screened reference genes. These results can provide more accurate and reliable qRT-PCR normalization for future studies on the expression patterns of genes regulating plant architecture of chrysanthemums.

Klíčová slova:

Gene expression – Gene regulation – Genetic screens – Gravitropism – Melting – Polymerase chain reaction – RNA synthesis – Transcriptome analysis


Zdroje

1. Udvardi MK, Czechowski T, Scheible WR (2008) Eleven golden rules of quantitative RT-PCR. Plant Cell 20: 1736–1737. doi: 10.1105/tpc.108.061143 18664613

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

3. 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 16166256

4. Hu Y, Fu H, Qiao H, Sun S, Zhang W, Jin S, et al. (2018) Validation and evaluation of reference genes for quantitative real-time PCR in Macrobrachium Nipponense. Int J Mol Sci 19: 2258.

5. Dheda K, Huggett JF, Chang J, Kim LU, Bustin SA, Johnson MA, et al. (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344: 141–143. doi: 10.1016/j.ab.2005.05.022 16054107

6. Wang H, Chen S, Jiang J, Zhang F, Chen F (2015) Reference gene selection for cross-species and cross-ploidy level comparisons in Chrysanthemum spp. Sci Rep 5: 8094. doi: 10.1038/srep08094 25627791

7. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6: 279–284. doi: 10.1038/sj.gene.6364190 15815687

8. Gu C, Chen S, Liu Z, Shan H, Luo H, Guan Z, et al. (2011) Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol 49: 192–197. doi: 10.1007/s12033-011-9394-6 21416201

9. Qi S, Yang L, Wen X, Hong Y, Song X, Zhang M, et al. (2016) Reference gene selection for RT-qPCR analysis of flower development in Chrysanthemum morifolium and Chrysanthemum lavandulifolium. Front Plant Sci 7: 287. doi: 10.3389/fpls.2016.00287 27014310

10. Anderson NO (2007) Flower breeding and genetics—issues, challenges and opportunities for the 21st century. 2nd ed. Springer-Verlag, New York, United States; 389–437.

11. Dai S, Wang W, Li M, Xu Y (2005) Phylogenetic relationship of Dendranthema (DC.) Des Moul. revealed by fluorescent in situ hybridization. J Integr Plant Biol 47: 783–791.

12. Huang D, Li X, Sun M, Zhang T, Pan H, Cheng T, et al. (2016) Identification and characterization of CYC-Like genes in regulation of ray floret development in Chrysanthemum morifolium. Front Plant Sci 7: 1633. doi: 10.3389/fpls.2016.01633 27872631

13. Liu H, Sun M, Du D, Pan H, Cheng T, Wang J, et al. (2016) Whole-transcriptome analysis of differentially expressed genes in the ray florets and disc florets of Chrysanthemum morifolium. BMC Genomics 17: 398. doi: 10.1186/s12864-016-2733-z 27225275

14. Wang P, Chen J (1990) Studies on breeding ground-cover chrysanthemum new cultivars. Acta Hortic Sin 8: 223–228.

15. Chen F, Fang W, Zhao H, Guan Z, Xu G (2005) New varieties of chrysanthemum-ground-cover varieties. Acta Hortic Sin 32: 1167.

16. Chen J, Zhong J, Shi X, Zhang Q, Sun M (2018) Chrysanthemum yantaiense, a rare new species of Asteraceae from China. Phytotaxa 374: 92.

17. Withers JC, Shipp MJ, Rupasinghe SG, Sukumar P, Schuler MA, Muday GK, et al. (2013) Gravity Persistent Signal 1 (GPS1) reveals novel cytochrome P450s involved in gravitropism. Am J Bot 100: 183–193. doi: 10.3732/ajb.1200436 23284057

18. Zhang N, Yu H, Yu H, Cai Y, Huang L, Xu C, et al. (2018) A core regulatory pathway controlling rice tiller angle mediated by the LAZY1-dependent asymmetric distribution of auxin. Plant Cell 30: 1461–1475. doi: 10.1105/tpc.18.00063 29915152

19. Hu L, Mei Z, Zang A, Chen H, Dou X, Jin J, et al. (2013) Microarray analyses and comparisons of upper or lower flanks of rice shoot base preceding gravitropic bending. PLoS ONE 8: e74646. doi: 10.1371/journal.pone.0074646 24040303

20. Li H, Chen X, Zhu F, Liu H, Hong Y, Liang X. (2013) Transcriptome profiling of peanut (Arachis hypogaea) gynophores in gravitropic response. Funct Plant Biol 40: 1249.

21. Zhang S, Chen S, Chen F, Teng N, Fang W, Guan Z. (2008) Anatomical structure and gravitropic response of the creeping shoots of ground-cover chrysanthemum ‘Yuhuajinhua’. Plant Growth Regul 56: 141–150.

22. Zhang S, Chen S, Chen F, Liu Z, Fang W. (2012) The regulatory role of the auxin in the creeping chrysanthemum habit. Russ J Plant Physiol 59: 364–371.

23. Xia S, Chen Y, Jiang J, Chen S, Guan Z, Fang W, et al. (2013) Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization. Mol Biol Rep 40: 237–246. doi: 10.1007/s11033-012-2054-5 23065216

24. Vandesompele J, de Preter K, Pattyn F, Poppe B, van Roy N, de Paepe A, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.

25. 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 15289330

26. 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. doi: 10.1023/b:bile.0000019559.84305.47 15127793

27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262 11846609

28. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8: R19. doi: 10.1186/gb-2007-8-2-r19 17291332

29. Li P, Wang Y, Qian Q, Fu Z, Wang M, Zeng D, et al. (2007) LAZY1 controls rice shoot gravitropism through regulating polar auxin transport. Cell Res 17: 402–410. doi: 10.1038/cr.2007.38 17468779

30. Yoshihara T, Iino M (2007) Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways. Plant Cell Physiol 48: 678–688. doi: 10.1093/pcp/pcm042 17412736

31. Dong Z, Jiang C, Chen X, Zhang T, Ding L, Song W, et al. (2013) Maize LAZY1 mediates shoot gravitropism and inflorescence development through regulating auxin transport, auxin signaling, and light response. Plant Physiol 163: 1306–1322. doi: 10.1104/pp.113.227314 24089437

32. Yoshihara T, Spalding EP, Iino M (2013) AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence. Plant J 74: 267–279. doi: 10.1111/tpj.12118 23331961

33. Taniguchi M, Furutani M, Nishimura T, Nakamura M, Fushita T, Iijima K, et al. (2017) The Arabidopsis LAZY1 family plays a key role in gravity signaling within statocytes and in branch angle control of roots and shoots. Plant Cell 29: 1984–1999. doi: 10.1105/tpc.16.00575 28765510

34. Yoshihara T, Spalding EP (2017) LAZY genes mediate the effects of gravity on auxin gradients and plant architecture. Plant Physiol 175: 959–969. doi: 10.1104/pp.17.00942 28821594

35. Noh B, Bandyopadhyay A, Peer WA, Spalding EP, Murphy AS (2003) Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1. Nature, 423: 999–1002. doi: 10.1038/nature01716 12827205

36. Wisniewska J, Xu J, Seifertova D, Brewer PB, Ruzicka K, Blilou I, et al. (2006) Polar PIN localization directs auxin flow in plants. Science 312: 883. doi: 10.1126/science.1121356 16601151

37. Remans T, Smeets K, Opdenakker K, Mathijsen D, Vangronsveld J, Cuypers A. (2008) Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta 227: 1343–1349. doi: 10.1007/s00425-008-0706-4 18273637

38. Fu J, Wang Y, Huang H, Zhang C, Dai S (2012) Reference gene selection for RT-qPCR analysis of Chrysanthemum lavandulifolium during its flowering stages. Mol Breeding 31: 205–215.

39. Hong Y, Dai S (2015) Selection of reference genes for real-time quantitative polymerase chain reaction analysis of light-dependent anthocyanin biosynthesis in chrysanthemum. J Am Soc Hortic Sci 140: 68–77.

40. Liu Y, Liu J, Xu L, Lai H, Chen Y, Yang Z, et al. (2017) Identification and validation of reference genes for seashore paspalum response to abiotic stresses. Int J Mol Sci 18: 1322.

41. Zheng T, Chen Z, Ju Y, Zhang H, Cai M, Pan H, et al. (2018) Reference gene selection for qRT-PCR analysis of flower development in Lagerstroemia indica and L. speciosa. PLoS One 13: e0195004. doi: 10.1371/journal.pone.0195004 29579116

42. Yeap WC, Loo JM, Wong YC, Kulaveerasingam H (2013) 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 Organ Cult 116: 55–66.

43. Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, et al. (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. doi: 10.1111/j.1467-7652.2008.00346.x 18433420

44. Lee JM, Roche JR, Donaghy DJ, 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. doi: 10.1186/1471-2199-11-8 20089196

45. Martin JA, Wang Z (2011) Next-generation transcriptome assembly. Nat Rev Genet 12: 671–682. doi: 10.1038/nrg3068 21897427

46. Schmidt GW, Delaney SK (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: 233–241. doi: 10.1007/s00438-010-0511-1 20098998

47. Xiao Z, Sun X, Liu X, Li C, He L, Chen S, et al. (2016) Selection of reliable reference genes for gene expression studies on Rhododendron molle G. Don. Front Plant Sci 7: 1547. doi: 10.3389/fpls.2016.01547 27803707

48. Li J, Han J, Hu Y, Yang J (2016) Selection of reference genes for quantitative real-time PCR during flower development in tree peony (Paeonia suffruticosa Andr.). Front Plant Sci 7: 516. doi: 10.3389/fpls.2016.00516 27148337

49. Poteryaev D, Spang A (2005) A role of SAND-family proteins in endocytosis. Biochem Soc Trans 33: 606–608. doi: 10.1042/BST0330606 16042554

50. Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10: 4. doi: 10.1186/1471-2229-10-4 20056000

51. Mafra V, Kubo KS, Alves-Ferreira M, Ribeiro-Alves M, Stuart RM, Boava LP, et al. (2012) Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7: e31263. doi: 10.1371/journal.pone.0031263 22347455

52. Ye X, Zhang F, Tao Y, Song S, Fang J (2015) Reference gene selection for quantitative real-time PCR normalization in different cherry genotypes, developmental stages and organs. Sci Hortic 181: 182–188.

53. Janssens V, Goris J (2001) Protein phosphatase 2A: A highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling. Biochem J 353: 417–439. doi: 10.1042/0264-6021:3530417 11171037

54. 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. doi: 10.1186/1471-2229-10-49 20302670

55. Jin X, Fu J, Dai S, Sun Y, Hong Y (2013) Reference gene selection for qPCR analysis in cineraria developing flowers. Sci Hort 153: 64–70.

56. Punzo P, Ruggiero A, Possenti M, Nurcato R, Costa A, Morelli G, et al. (2018) The PP2A-interactor TIP41 modulates ABA responses in Arabidopsis thaliana. Plant J 94: 991–1009. doi: 10.1111/tpj.13913 29602224

57. Exposito-Rodriguez 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 19102748

58. Xue S, Zou J, Liu Y, Wang M, Zhang C, Le J (2019) Involvement of BIG5 and BIG3 in BRI1 Trafficking Reveals Diverse Functions of BIG-subfamily ARF-GEFs in Plant Growth and Gravitropism. Int J Mol Sci 20: 2339.


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2019 Číslo 11