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Journal of Applied Phycology

, Volume 31, Issue 1, pp 787–793 | Cite as

Selection of reference genes for real-time RT-PCR normalization in brown alga Undaria pinnatifida

  • Jing Li
  • Haishu Huang
  • Tifeng Shan
  • Shaojun PangEmail author
Article

Abstract

Quantitative real-time PCR (qRT-PCR) has become widely used method for detecting gene expression of an organism at different developmental stages or under various stressful environmental conditions. Finding reference genes is a prerequisite in this process. In this research, we investigated the expression stability of eight candidate genes in the brown alga Undaria pinnatifida, including EF1α (elongation factor 1-alpha), eEF1β (eukaryotic elongation factor-1 B beta), RPL35 (60S ribosomal protein L35-3), GAPDH (glyceraldehyde-3-phosphatedehydrogenase), RPS17 (40S ribosomal protein S17), LHCP (light-harvesting complex protein), ribH (6,7-dimethyl-8-ribityllumazine synthase), and ilvC (ketol-acid reductoisomerase). The haploid male and female gametophytes at different stages, as well as the diploid sporophytes treated with different abiotic factors were analyzed by geNorm and NormFinder software. It was found that eEF1β and ribH could be used as the most stable reference genes in studies of gender or developmental gene expression in the gametophytes. As for the detection of gene expression in the sporophytes, eEF1β and ribH were recommended as the most suitable reference genes for irradiance treatment, eEF1β and RPL35 for temperature treatment, while EF1α and LHCP for nutrition treatment. These results will enable us to go further for quantifying gene expression study at transcript level in U. pinnatifida.

Keywords

Reference genes qRT-PCR geNorm NormFinder Phaeophyta Undaria pinnatifida 

Notes

Funding information

This research was supported by projects from the National Natural Science Foundation of China (No. 41476141, No. 31702365, and No. 41676128) and the Sino-German Science Center (GZ 1080), China Agriculture Research System (CARS-50), National Key Technology Support Program (2015BAD13B05), the National Infrastructure of Fishery Germplasm Resource (2017DKA30470), the Taishan Scholar Program of Shandong Province, and Huiquan Scholar Program of Institute of Oceanology, Chinese Academy of Sciences.

References

  1. Alves-Lima C, Cavacana N, Teixeira Chaves GA, de Lima NO, Stefanello E, Colepicolo P, Hotta CT (2017) Reference genes for transcript quantification in Gracilaria tenuistipitata under drought stress. J Appl Phycol 29:731–740CrossRefGoogle Scholar
  2. 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 64:5245–5250CrossRefGoogle Scholar
  3. Apt KE, Clendennen SK, Powers DA, Grossman AR (1995) The gene family encoding the fucoxanthin chlorophyll proteins from the brown alga Macrocystis pyrifera. Mol Gen Genet 246:455–464CrossRefGoogle Scholar
  4. Artico S, Nardeli SM, Neto OBO, 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 10:49CrossRefGoogle Scholar
  5. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Molec Endocrinol 34:597–601CrossRefGoogle Scholar
  6. Casas GN, Piriz ML, Parodi ER (2008) Population features of the invasive kelp Undaria pinnatifida (Phaeophyceae: Laminariales) in Nuevo Gulf (Patagonia, Argentina). J Mar Biol Assoc U K 88:21–28CrossRefGoogle Scholar
  7. 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 139:5–17CrossRefGoogle Scholar
  8. Henkel S, Hofmann G (2008) Differing patterns of hsp70 gene expression in invasive and native kelp species: evidence for acclimation-induced variation. J Appl Phycol 20:915–924CrossRefGoogle Scholar
  9. Henkel SK, Kawai H, Hofmann GE (2009) Interspecific and interhabitat variation in hsp70 gene expression in native and invasive kelp populations. Mar Ecol Prog Ser 386:1–13CrossRefGoogle Scholar
  10. Jensen J, Ørntoft T (2004) Normalization of real-time quantitative RT-PCR data: a model based variance estimation approach to identify genes suited for normalization-applied to bladder-and colon-cancer data-sets. Cancer Res 64:5245–5250CrossRefGoogle Scholar
  11. Jiang Q, Xu ZS, Wang F, Li MY, Ma J, Xiong AS (2014) Effects of abiotic stresses on the expression of Lhcb1 gene and photosynthesis of Oenanthe javanica and Apium graveolens. Biol Plantarum 58:256–264CrossRefGoogle Scholar
  12. Kanda T (1936) On the gametophyte of some Japanese species of Laminariales. Sci Pap Inst Algol Res Fac Sci Hokkaido Univ 1:221–260Google Scholar
  13. Kong F, Cao M, Sun P, Liu W, Mao Y (2015) Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR. J Appl Phycol 27:1003–1010CrossRefGoogle Scholar
  14. Kowalczyk N, Rousvoal S, Herve C, Boyen C, Collen J (2014) RT-qPCR normalization genes in the red alga Chondrus crispus. PLoS One 9:e0086574CrossRefGoogle Scholar
  15. Le Bail A, Dittami SM, de Franco P-O, Rousvoal S, Cock MJ, Tonon T, Charrier B (2008) Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Molec Biol 9:75CrossRefGoogle Scholar
  16. Li J, Pang S, Shan T, Liu F, Gao S (2014) Zoospore-derived monoecious gametophytes in Undaria pinnatifida (Phaeophyceae). Chin J Oceanol Limnol 32:365–371CrossRefGoogle Scholar
  17. Li J, Pang S, Shan T (2017) Existence of an intact male life cycle offers a novel way in pure-line crossbreeding in the brown alga Undaria pinnatifida. J Appl Phycol 29:993–999CrossRefGoogle Scholar
  18. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914CrossRefGoogle Scholar
  19. Pattyn F, Speleman F, De Paepe A, Vandesompele J (2003) RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res 31:122–123CrossRefGoogle Scholar
  20. Primo C, Hewitt CL, Campbell ML (2010) Reproductive phenology of the introduced kelp Undaria pinnatifida (Phaeophyceae, Laminariales) in Port Phillip Bay (Victoria, Australia). Biol Invas 12:3081–3092CrossRefGoogle Scholar
  21. Provasoli L. 1968. Media and prospects for the cultivation of marine algae. Proceedings of the US-Japan Conference, Hakone, 12–15 September 1966, pp 63–75Google Scholar
  22. Shan TF, Pang SJ, Li J, Li X. 2015. De novo transcriptome analysis of the gametophyte of Undaria pinnatifida (Phaeophyceae). J Appl Phycol 27: 1011–1019Google Scholar
  23. Shim J, Shim E, Kim GH, Han JW, Zuccarello GC (2016) Keeping house: evaluation of housekeeping genes for real-time PCR in the red alga, Bostrychia moritziana (Florideophyceae). Algae 31:167–174CrossRefGoogle Scholar
  24. 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(7):0034CrossRefGoogle Scholar
  25. Wu X, Huang A, Xu M, Wang C, Jia Z, Wang G, Niu J (2013) Variation of expression levels of seven housekeeping genes at different life-history stages in Porphyra yezoensis. PLoS One 8(4):e60740CrossRefGoogle Scholar
  26. Zhao P, Wang K, Zhang W, Liu HY, Du LP, Hu HR, Ye XG (2017) Comprehensive analysis of differently expressed genes and proteins in albino and green plantlets from a wheat anther culture. Biol Plantarum 61:255–265CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.CAS Key Laboratory of Experimental Marine Biology, Institute of OceanologyChinese Academy of SciencesQingdaoPeople’s Republic of China
  2. 2.Laboratory for Marine Biology and BiotechnologyQingdao National Laboratory for Marine Science and TechnologyQingdaoPeople’s Republic of China
  3. 3.University of Chinese Academy of SciencesBeijingChina

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