Molecular Breeding

, Volume 34, Issue 3, pp 1437–1447

The first Illumina-based de novo transcriptome sequencing and analysis of pumpkin (Cucurbita moschata Duch.) and SSR marker development

  • Tingquan Wu
  • Shaobo Luo
  • Rui Wang
  • Yujuan Zhong
  • Xiaomei Xu
  • Yu’e Lin
  • Xiaoming He
  • Baojuan Sun
  • Hexun Huang
Article

Abstract

Pumpkin (Cucurbita moschata Duch.) is an important vegetable crop cultivated worldwide. In this study, the pumpkin transcriptome was sequenced by RNA-seq using the Illumina Hiseq 2000. A total of 52,849,316 clean sequencing reads, 66,621 contigs and 62,480 unigenes were postulated. Based on similarity searches with known proteins, 47,899 genes (76.66 % of the unigenes) were annotated: 47,596, 34,368 and 16,700 mapped in Nr, Swissprot and COG classifications, respectively; 21,164 were annotated with 44 gene ontology functional categories; and 13,728 were annotated to 269 pathways by searching the Kyoto Encyclopedia of Genes and Genomes pathway database. A total of 7,814 simple sequence repeats (SSRs) were identified in these unigenes and 4,794 pairs of primers were designed for application of SSRs. To date, 35 SSRs have been validated in 12 pumpkin varieties and can separate the pumpkin varieties into Cucurbita maxima and Cucurbita moschata. In addition, the expression of eight photoperiod-related unigenes were studied in different pumpkin plants and it was deduced that they may contribute to late flowering and light insensitiveness. This research will provide an important platform to facilitate gene discovery for functional genome studies of pumpkin and to conduct SSR discovery for breeders for use in pumpkin breeding.

Keywords

Pumpkin Transcriptome SSR primers Illumina sequencing PPIS pumpkin plants 

Abbreviations

GO

Gene ontology

KEGG

The Kyoto Encyclopedia of Genes and Genomes pathway database

SSR

Simple sequence repeat

PPIS

Photoperiod-insensitive

PPS

Photoperiod-sensitive

FAO

Food and Agriculture Organization of the United Nations

CDS

Coding sequences

Supplementary material

11032_2014_128_MOESM1_ESM.tif (23.5 mb)
Supplementary material 1 (TIFF 24106 kb)
11032_2014_128_MOESM2_ESM.tif (43.4 mb)
Supplementary material 2 (TIFF 44442 kb)
11032_2014_128_MOESM3_ESM.tif (40.8 mb)
Supplementary material 3 (TIFF 41747 kb)
11032_2014_128_MOESM4_ESM.tif (72.6 mb)
Supplementary material 4 (TIFF 74299 kb)
11032_2014_128_MOESM5_ESM.tif (32.5 mb)
Supplementary material 5 (TIFF 33248 kb)
11032_2014_128_MOESM6_ESM.tif (70.3 mb)
Supplementary material 6 (TIFF 71937 kb)
11032_2014_128_MOESM7_ESM.tif (22.4 mb)
Supplementary material 7 (TIFF 22945 kb)
11032_2014_128_MOESM8_ESM.xlsx (24 kb)
Supplementary material 8 (XLSX 25 kb)
11032_2014_128_MOESM9_ESM.docx (16 kb)
Supplementary material 9 (DOCX 17 kb)
11032_2014_128_MOESM10_ESM.xlsx (1.2 mb)
Supplementary material 10 (XLSX 1273 kb)
11032_2014_128_MOESM11_ESM.xlsx (1.1 mb)
Supplementary material 11 (XLSX 1119 kb)
11032_2014_128_MOESM12_ESM.docx (18 kb)
Supplementary material 12 (DOCX 19 kb)
11032_2014_128_MOESM13_ESM.docx (13 kb)
Supplementary material 13 (DOCX 13 kb)
11032_2014_128_MOESM14_ESM.xlsx (15 kb)
Supplementary material 14 (XLSX 16 kb)
11032_2014_128_MOESM15_ESM.docx (19 kb)
Supplementary material 15 (DOCX 20 kb)
11032_2014_128_MOESM16_ESM.docx (19 kb)
Supplementary material 16 (DOCX 19 kb)

References

  1. Ando K, Carr KM, Grumet R (2012) Transcriptome analyses of early cucumber fruit growth identifies distinct gene modules associated with phases of development. BMC Genom 13:518CrossRefGoogle Scholar
  2. Blanca J, Esteras C, Ziarsolo P, Pérez D, Fernández-Pedrosa V, Collado C et al (2012) Transcriptome sequencing for SNP discovery across Cucumis melo. BMC Genom 13:280CrossRefGoogle Scholar
  3. 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 21:3674–3676PubMedCrossRefGoogle Scholar
  4. Fabbrini F, Gaudet M, Bastien C, Zaina G, Harfouche A, Beritognolo I et al (2012) Phenotypic plasticity, QTL mapping and genomic characterization of bud set in black poplar. BMC Plant Biol 12:47PubMedCrossRefPubMedCentralGoogle Scholar
  5. Garcia-Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, González MV et al (2012) The genome of melon (Cucumis melo L.). Proc Natl Acad Sci USA 109:11872–11877PubMedCrossRefPubMedCentralGoogle Scholar
  6. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652PubMedCrossRefPubMedCentralGoogle Scholar
  7. Guo SG, Liu JG, Zheng Y, Huang MY, Zhang HY, Gong GY et al (2011) Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles. BMC Genom 12:454CrossRefGoogle Scholar
  8. Guo SG, Zhang JG, Sun HH, Salse J, Lucas WJ, Zhang HY et al (2012) The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45:51–58PubMedCrossRefGoogle Scholar
  9. Hall A, Bastow RM, Davis SJ, Hanano S, McWatters HG, Hibberd V (2003) The TIME FOR COFFEE gene maintains the amplitude and timing of Arabidopsis circadian clocks. Plant Cell 15:2719–2729PubMedCrossRefPubMedCentralGoogle Scholar
  10. Huang SW, Li RQ, Zhang ZH, Li L, Gu XF, Fan W et al (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet 41:1275–1281PubMedCrossRefGoogle Scholar
  11. Huang LL, Yang X, Sun P, Tong W, Hu SQ (2012) The first Illumina-based de novo transcriptome sequencing and analysis of safflower flowers. PLoS ONE 7:e38653CrossRefGoogle Scholar
  12. Hyun TK, Rim Y, Jang HJ, Kim CH, Park J, Kumar R et al (2012) De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis. Plant Mol Biol 79(4–5):413–427PubMedCrossRefGoogle Scholar
  13. Iseli, C., Jongeneel, C.V., Bucher, P. (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc Int Conf Intell Syst Mol Biol: 138–148Google Scholar
  14. Isutsa DK, Mallowa SO (2013) Increasing leaf harvest intensity enhances edible leaf vegetable yields and decreases mature fruit yields in multi-purpose pumpkin. J Agric Biol Sci 8:610–615Google Scholar
  15. Ito S, Song YH, Josephson-Day AR, Miller RJ, Breton G, Olmstead RG (2012) FLOWERING BHLH transcriptional activators control expression of the photoperiodic flowering regulator CONSTANS in Arabidopsis. Proc Natl Acad Sci USA 109:3582–3587PubMedCrossRefPubMedCentralGoogle Scholar
  16. Jiang B, Xie DS, Liu WR, Peng QW, He XM (2013) De Novo assembly and characterization of the transcriptome, and development of SSR Markers in wax gourd (Benicasa hispida). PLoS ONE 8(8):e71054PubMedCrossRefPubMedCentralGoogle Scholar
  17. Kim J, Kim Y, Yeom M, Kim JH, Nam HG (2008) FIONA1 is essential for regulating period length in the Arabidopsis circadian clock. Plant Cell 20:307–319PubMedCrossRefPubMedCentralGoogle Scholar
  18. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformation 24:713–714CrossRefGoogle Scholar
  19. Lu TT, Lu GJ, Fan DL, Zhu CR, Li W, Zhao Q et al (2010) Function annotation of the rice transcriptome at single-nucleotide resolution by RNA-seq. Genome Res 20:1238–1249PubMedCrossRefPubMedCentralGoogle Scholar
  20. Morozova O, Marra MA (2008) Applications of next-generation sequencing technologies in functional genomics. Genomics 92:255–264PubMedCrossRefGoogle Scholar
  21. Murray MG, Thompson WF (1980) Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefPubMedCentralGoogle Scholar
  22. Neeraja CN, Maghirang-Rodriguez R, Pamplona A, Heuer S, Collard BC et al (2007) A marker-assisted backcross approach for developing submergence-tolerant rice cultivars. Theor Appl Genet 115:767–776PubMedCrossRefGoogle Scholar
  23. Noh YS, Amasino RM (2003) PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis. Plant Cell 15:1671–1682PubMedCrossRefPubMedCentralGoogle Scholar
  24. Noh B, Lee SH, Kim HJ, Yi G, Shin EA, Lee M et al (2004) Divergent roles of a pair of homologous Jumonji/zinc-finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16:2601–2613PubMedCrossRefPubMedCentralGoogle Scholar
  25. Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS et al (1999) Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285:1579–1582PubMedCrossRefGoogle Scholar
  26. Sato E, Nakamichi N, Yamashino T, Mizuno T (2002) Aberrant expression of the Arabidopsis circadian-regulated APRR5 gene belonging to the APRR1/TOC1 quintet results in early flowering and hypersensitiveness to light in early photomorphogenesis. Plant Cell Physiol 43:1374–1385PubMedCrossRefGoogle Scholar
  27. Senior ML, Murphy JP, Goodman MM, Stuber CW (1998) Utility of SSRs for determining genetic similarities and relationships in maize using an agarose gel system. Crop Sci 38:1088–1098CrossRefGoogle Scholar
  28. Temnykh S, DeClerck G, Lukashova A, Lipovich L, Cartinhour S, Lipovich L et al (2001) Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential. Genome Res 11:1441–1452PubMedCrossRefPubMedCentralGoogle Scholar
  29. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G et al (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515PubMedCrossRefPubMedCentralGoogle Scholar
  30. Tseng TS, Salomé PA, McClung CR, Olszewski NE (2004) SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in light responses, flowering, and rhythms in cotyledon movements. Plant Cell 16:1550–1563PubMedCrossRefPubMedCentralGoogle Scholar
  31. Wang P, Liu JC, Zhao QY (2002) Studies on nutrient composition and utilization of pumpkin fruit. J Inner Mongolia Agric Univ 23:52–54Google Scholar
  32. Wang ZY, Fang BP, Chen JY, Zhang XJ, Luo ZX, Huang LF et al (2010) De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas). BMC Genom 11:726CrossRefGoogle Scholar
  33. Wu JF, Wang Y, Wu SH (2008) Two new clock proteins, LWD1 and LWD2, regulate Arabidopsis photoperiodic flowering. Plant Physiol 148:948–959PubMedCrossRefPubMedCentralGoogle Scholar
  34. Wu TQ, Tang DZ, Chen WD, Huang HH, Wang R, Chen YF (2013) Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria. Gene 527:235–242PubMedCrossRefGoogle Scholar
  35. Yadav M, Jain S, Tomar R, Prasad GBKS, Yadav H (2010) Medicinal and biological potential of pumpkin: an updated review. Nutr Res Rev 23:184–190PubMedCrossRefGoogle Scholar
  36. Ye J, Fang L, Zheng HK, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:293–297CrossRefGoogle Scholar
  37. Yu X, Li L, Li L, Guo M, Chory J, Yin Y (2008) Modulation of brassinosteroid-regulated gene expression by Jumonji domain-containing proteins ELF6 and REF6 in Arabidopsis. Proc Natl Acad Sci USA 105:7618–7623PubMedCrossRefPubMedCentralGoogle Scholar
  38. Zhang F, Jiang ZM, Zhang EM (2000) Pumpkin function properties and application in food industry. Sci Technol Food Indus 21:62–64Google Scholar
  39. Zhang JN, Liang S, Duan JL, Wang J, Chen SL, Cheng ZS et al (2012) De novo assembly and characterization of the transcriptome during seed development, and generation of genic-SSR markers in Peanut (Arachis hypogaea L.). BMC Genom 13:90CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Tingquan Wu
    • 1
    • 2
  • Shaobo Luo
    • 1
    • 2
  • Rui Wang
    • 1
    • 2
  • Yujuan Zhong
    • 1
    • 2
  • Xiaomei Xu
    • 1
    • 2
  • Yu’e Lin
    • 1
  • Xiaoming He
    • 1
    • 2
  • Baojuan Sun
    • 1
    • 2
  • Hexun Huang
    • 1
  1. 1.Vegetable Research InstituteGuangdong Academy of Agricultural SciencesGuangzhouChina
  2. 2.Guangdong Key Laboratory for New Technology Research of VegetablesGuangzhouChina

Personalised recommendations