Skip to main content
Log in

Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The tuberous root of radish is an important vegetable, but insufficient transcriptomic and genomic data are currently available to understand the molecular mechanisms underlying tuberous root formation and development. High-throughput transcriptomic sequencing is essential to generate a large transcript sequence data set for gene discovery and molecular marker development. In this study, a total of 107.3 million clean reads were generated using Illumina paired-end sequencing technology. De novo assembly generated 61,554 unigenes with an average length of 820 bp. Based on a sequence similarity search with known proteins or nucleotides, 85.51 % (52,634), 90.18 % (55,507) and 54 % (33,242) consensus sequences showed homology with sequences in the Nr, Nt and Swiss-Prot databases, respectively. Of these annotated unigenes, 21,109 and 17,343 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. A total of 27,809 unigenes were assigned to 123 pathways in the Kyoto Encyclopedia of Genes and Genomes database. Analysis of transcript differences between libraries from the early and late seedling developmental stages demonstrated that starch and sucrose metabolism and phenylpropanoid biosynthesis may be the dominant metabolic events during tuberous root formation and plant hormones probably play critical roles in regulation of this developmental process. In total, 14,641 potential EST-SSRs were identified among the unigenes, and 12,733 primer pairs for 2,511 SSR were obtained. Summarily, this study gave us a clue to understand the radish tuberous root formation and development, and also provided us with a valuable sequence resource for novel gene discovery and marker-assisted selective breeding in radish.

Key message

De novo assembled and characterized the radish tuberous root transcriptome; explored the mechanism of radish tuberous root formation; development of EST-SSR markers in radish.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Audic S, Claverie JM (1997) The significance of digital gene expression profiles. Genome Res 7:986–995

    PubMed  CAS  Google Scholar 

  • Barakat A, DiLoreto DS, Zhang Y, Smith C, Baier K, Powell WA, Wheeler N, Sederoff R, Carlson JE (2009) Comparison of the transcriptomes of American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) in response to the chestnut blight infection. BMC Plant Biol 9:51

    Article  PubMed  Google Scholar 

  • Benjamini Y, Yekutieli D (2001) The control of the false discovery rate in multiple testing under dependency. Ann Statist 29:1165–1188

    Article  Google Scholar 

  • Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2011) GenBank. Nucleic Acids Res 39:32–37

    Article  Google Scholar 

  • Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, Nusbaum C, Jaffe DB (2008) ALLPATHS de novo assembly of whole-genome shotgun microreads. Genome Res 18(5):810–820

    Article  PubMed  CAS  Google Scholar 

  • Cameron M, Williams HE, Cannane A (2004) Improved gapped alignment in BLAST. IEEE/ACM Trans Comput Biol Bioinforma 1:116–129

    Article  CAS  Google Scholar 

  • Collins LJ, Biggs PJ, Voelckel C, Joly S (2008) An approach to transcriptome analysis of non-model organisms using short-read sequences. Genome Inform 21:3–14

    Article  PubMed  CAS  Google Scholar 

  • Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18):3674–3676

    Article  PubMed  CAS  Google Scholar 

  • Curtis IS (2003) The noble radish: past, present and future. Trends Plant Sci 8:305–307

    Article  PubMed  CAS  Google Scholar 

  • Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotech 29:644–652

    Article  CAS  Google Scholar 

  • Gupta AK, Singh J, Kaur N (2001) Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.). Acta Physiol Plant 23:31–36

    Article  CAS  Google Scholar 

  • Hale MC, McCormick CR, Jackson JR, Dewoody JA (2009) Next-generation pyrosequencing of gonad transcriptomes in the polyploid lake sturgeon (Acipenser fulvescens): the relative merits of normalization and rarefaction in gene discovery. BMC Genomics 10:203

    Article  PubMed  Google Scholar 

  • Hegedus Z, Zakrzewska A, Agoston VC, Ordas A, Racz P, Mink M, Spaink HP, Meijer AH (2009) Deep sequencing of the zebrafish transcriptome response to mycobacterium infection. Mol Immunol 46(15):2918–2930

    Article  PubMed  CAS  Google Scholar 

  • Huang YW, Huang TH, Wang LS (2010) Profiling DNA methylomes from microarray to genome-scale sequencing. Technol Cancer Res Treat 9:139–147

    PubMed  CAS  Google Scholar 

  • Jakmatakul R, Suttisri R, Tengamnuay P (2009) Evaluation of antityrosinase and antioxidant activities of Raphanus sativus root: comparison between freeze-dried juice and methanolic extract. Thai J Pharm Sci 33:22–30

    Google Scholar 

  • Johnston JS, Pepper AE, Hall AE, Chen ZJ, Hodnett G, Drabek J, Lopez R, Price HJ (2005) Evolution of genome size in Brassicaceae. Ann Bot 95(1):229–235

    Article  PubMed  CAS  Google Scholar 

  • Kantety RV, La Rota M, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48:501–510

    Article  PubMed  CAS  Google Scholar 

  • Kortt AA, Caldwell JB, Lilley GG, Higgins TJV (1991) Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.). Eur J Biochem 195(2):329–334

    Article  PubMed  CAS  Google Scholar 

  • Kumpatla SP, Mukhopadhyay S (2005) Mining and survey of simple sequence repeats in expressed sequence tags of dicotyledonous species. Genome 48(6):985–998

    Article  PubMed  CAS  Google Scholar 

  • Li JW, Lu ZH, Wang RY, Lu GY, Ge XG, Zhang Y, Chen JF, Jiang XM (1979) Cultivation of vegetables. Agricultural Press, Beijing (in Chinese)

    Google Scholar 

  • Li R, Fan W, Tian G, Zhu H, He L, Cai J, Huang Q, Cai Q, Li B, Bai Y et al (2010a) The sequence and de novo assembly of the giant panda genome. Nature 463:311–317

    Article  PubMed  CAS  Google Scholar 

  • Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K et al (2010b) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 20(2):265–272

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Lu Z, Liu L, Li X, Gong Y, Hou X, Zhu X, Yang J, Wang L (2008) Analysis and evaluation of nutritional quality in Chinese radish (Raphanus sativus L.). Agri Sci China 7(7):823–830

    Article  CAS  Google Scholar 

  • Meyer E, Aglyamova GV, Wang S, Buchanan-Carter J, Abrego D, Colbourne JK, Willis BL, Matz MV (2009) Sequencing and de novo analysis of a coral larval transcriptome using 454 GSFlx. BMC Genomics 10:219

    Article  PubMed  Google Scholar 

  • Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30(2):194–200

    Article  PubMed  CAS  Google Scholar 

  • Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628

    Article  PubMed  CAS  Google Scholar 

  • Parchman TL, Geist KS, Grahnen JA, Benkman CW, Buerkle CA (2010) Transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery. BMC Genomics 11:180

    Article  PubMed  Google Scholar 

  • Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B et al (2003) TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19(5):651–652

    Article  PubMed  CAS  Google Scholar 

  • Powell W, Machray GC, Provan J (1996) Polymorphism revealed by simple sequence repeats. Trends Plant Sci 1:215–222

    Google Scholar 

  • Prat S, Frommer WB, Höfgen R, Keil M, Koßmann J, Köster-Töpfer M, Liu XJ, Müller B, Peña-Cortés H, Rocha-Sosa M et al (1990) Gene expression during tuber development in potato plants. FEBS Lett 268(2):334–338

    Article  PubMed  CAS  Google Scholar 

  • Rouhier H, Usuda H (2001) Spatial and temporal distribution of sucrose synthase in the radish hypocotyl in relation to thickening growth. Plant Cell Physiol 42(6):583–593

    Article  PubMed  CAS  Google Scholar 

  • Rubatzky VE, Yamaguchi M (1997) World vegetables: principles, production, and nutritive values, 2nd edn. Chapman & Hall, USA

    Google Scholar 

  • Shirasawa K, Oyama M, Hirakawa H, Sato S, Tabata S, Fujioka T, Kimizuka-Takagi C, Sasamoto S, Watanabe A, Kato M et al (2011) An EST-SSR linkage map of Raphanus sativus and comparative genomics of the Brassicaceae. DNA Res 18:221–232

    Article  PubMed  CAS  Google Scholar 

  • Squirrell J, Hollingsworth PM, Woodhead M, Russell J, Lowe AJ, Gibby M, Powell W (2003) How much effort is required to isolate nuclear microsatellites from plants? Mol Ecol 12(6):1339–1348

    Article  PubMed  CAS  Google Scholar 

  • Sun C, Li Y, Wu Q, Luo H, Sun Y, Song J, Lui EM, Chen S (2010) De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis. BMC Genomics 11:262

    Article  PubMed  Google Scholar 

  • Toth G, Gaspari Z, Jurka J (2000) Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res 10(7):967–981

    Article  PubMed  CAS  Google Scholar 

  • Trick M, Long Y, Meng J, Bancroft I (2009) Single nucleotide polymorphism (SNP) discovery in the polyploid Brassica napus using Solexa transcriptome sequencing. Plant Biotechnol J 7(4):334–346

    Article  PubMed  CAS  Google Scholar 

  • Tsuro M, Suwabe K, Kubo N, Matsumoto S, Hirai M (2008) Mapping of QTLs controlling root shape and red pigmentation in radish, Raphanus sativus L. Breed Sci 58:55–61

    Article  CAS  Google Scholar 

  • Usuda H, Demura T, Shimogawara K, Fukuda H (1999) Development of sink capacity of the “storage root” in a radish cultivar with a high ratio of “storage root” to shoot. Plant Cell Physiol 40(4):369–377

    Article  CAS  Google Scholar 

  • Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotech 23(1):48–55

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Wang B, Guo G, Wang C, Lin Y, Wang X, Zhao M, Guo Y, He M, Zhang Y, Pan L (2010a) Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing. Nucleic Acids Res 38(15):5075–5087

    Article  PubMed  CAS  Google Scholar 

  • Wang XW, Luan JB, Li JM, Bao YY, Zhang CX, Liu SS (2010b) De novo characterization of a whitefly transcriptome and analysis of its gene expression during development. BMC Genomics 11:400

    Article  PubMed  Google Scholar 

  • Wang Z, Fang B, Chen J, Zhang X, Luo Z, Huang L, Chen X, Li Y (2010c) De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas). BMC Genomics 11:726

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039

    Article  PubMed  CAS  Google Scholar 

  • Wei W, Qi X, Wang L, Zhang Y, Hua W, Li D, Lv H, Zhang X (2011) Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers. BMC Genomics 12:451

    Article  PubMed  CAS  Google Scholar 

  • Werth E (1937) Abstammung und Heimat des Rettichs. Angew Bot 19:194–205

    Google Scholar 

  • Wu T, Qin Z, Zhou X, Feng Z, Du Y (2010) Transcriptome profile analysis of floral sex determination in cucumber. J Plant Physiol 167(11):905–913

    Google Scholar 

  • Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation: a review. Mol Ecol 11(1):1–16

    Article  PubMed  CAS  Google Scholar 

  • Zeng S, Xiao G, Guo J, Fei Z, Xu Y, Roe B, Wang Y (2010) Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimedium sagittatum (Sieb. Et Zucc.) Maxim. BMC Genomics 11(1):94

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Modern Agricultural Industrial Technology System Funding of Shandong Province, China (2010sdxdcyjstxshucai); National Nature Science Foundation of China (31101553); Prospect of Shandong Seed Project, China (2011lzgcshucaizy) and the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province, China (BS2010SW027).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Jianwei Gao or Fengde Wang.

Additional information

Communicated by W. Harwood.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, S., Wang, X., He, Q. et al. Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish. Plant Cell Rep 31, 1437–1447 (2012). https://doi.org/10.1007/s00299-012-1259-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-012-1259-3

Keywords

Navigation