PDF herunterladen
Plant Biol (Stuttg) 2002; 4(6): 700-710
DOI: 10.1055/s-2002-37405
Original Paper
Georg Thieme Verlag Stuttgart ·New York

EST Sequencing, Annotation and Macroarray Transcriptome Analysis Identify Preferentially Root-Expressed Genes in Sugar Beet

D. Bellin 1 , M. Werber 1 , T. Theis 2 , B. Schulz 3 , B. Weisshaar 1,2 , K. Schneider 1
  • 1 Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Köln, Germany
  • 2 ADIS unit at Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Köln, Germany
  • 3 KWS SAAT AG, Grimsehlstraße 31, 37555 Einbeck, Germany
Weitere Informationen

Publikationsverlauf

Received: July 15, 2002

Accepted: November 14, 2002

Publikationsdatum:
24. Februar 2003 (online)

   Lizenzen und Reprints

Abstract

An integrated approach involving EST sequencing, data mining and multiparallel expression profiling by macroarray analysis was established to classify sugar beet gene products with respect to their expression in three different organs. The DNA sequences of 2996 ESTs derived from young sugar beet plants specified 2048 unique gene products with putative functions in primary and secondary metabolism (651), transport processes (136), signal transduction (78) and cellular organization (39). The cDNA clone collection was the basis for the generation of a macroarray. Sensitivity and reproducibility of our macroarray hybridization procedure were estimated first. The detection limit was found to correspond to 10 - 50 copies of single transcripts per cell. Within an interval of two-fold variation in signal intensities, reproducibility between spots on the same filter was determined to be 98.9 %, between spots on different filters 89.8 %, and reproducibility after hybridization with two probes synthesized from the same poly(A)+RNA sample was 97.6 %. Expression profiles from roots, leaves and inflorescences of field-grown plant material were generated. Two different samples of each organ were analysed to reduce sampling effects, which accounted on average for 30.3 % of spots with at least two-fold deviation. Expression values for each organ were determined by a stringent statistical evaluation of eight hybridizations for each clone. Macroarray expression data were confirmed by Northern blot analysis and quantitative RT-PCR experiments concerning eleven cDNAs. The analysis was then focused on 76 unique cDNAs, for which the amount of detected transcript in roots was at least twice as high as in the other organs tested. Functions of preferentially root-expressed candidate genes in taproot morphology and physiology are discussed.

Key words

EST - cluster analysis - macroarray - expression profiling - sugar beet - root-expressed genes

Reference

  • 01 Aharoni,  A.,, Keizer,  L. C. P.,, Van den Broeck,  H. C.,, Blanco-Portales,  R.,, Munoz-Blanco,  J.,, Bois,  G.,, Smit,  P.,, De Vos,  R. C. H.,, and O'Connell,  A. P.. (2002);  Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit.  Plant Phys.. 129 1019-1031
    Google Scholar
  • 02 Aharoni,  A., and Vorst,  O.. (2002);  DNA microarrays for functional plant genomics.  Plant Mol. Biol.. 48 99-118
    Google Scholar
  • 03 Altschul,  S. F.,, Gish,  W.,, Miller,  W.,, Myers,  E. W.,, and Lipman,  D. J.. (1990);  Basic local alignment search tool.  J. Mol. Biol.. 215 403-410
    Google Scholar
  • 04 Bartels,  D.,, Altosaar,  I.,, Harberd,  N. P.,, Barker,  R. F.,, and Thompson,  R. D.. (1986);  Molecular analysis of gliadin gene families at the complex Gli-1 locus of bread wheat (T. aestivum L.).  Theor. Appl. Genet.. 72 845-853
    Google Scholar
  • 05 Bartels,  D., and Thompson,  R. D.. (1983);  The characterization of cDNA clones coding for wheat storage proteins.  Nucleic Acids Res.. 11 2961-2978
    Google Scholar
  • 06 Bernard,  K.,, Auphan,  N.,, Granjeaud,  S.,, Victorero,  G.,, Schmitt-Verhulst,  A. M.,, Jordan,  B. R.,, and Nguyen,  C.. (1996);  Multiplex messenger assay: simultaneous, quantitative measurement of expression of many genes in the context of T-cell activation.  Nucleic Acids Res.. 24 1435-1442
    Google Scholar
  • 07 Burke,  J.,, Davison,  D.,, and Hide,  W.. (1999);  D2-cluster: A validated method for clustering EST and full-length cDNA sequences.  Genome Res.. 9 1135-1142
    Google Scholar
  • 08 Campbell,  P., and Braam,  J.. (1999);  In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis. .  The Plant Journal. 18 371-382
    Google Scholar
  • 09 Davies,  C., and Robinson,  S. P.. (2000);  Differential screening indicates a dramatic change in mRNA profiles during grape berry ripening. Cloning and characterization of cDNAs encoding putative cell wall and stress response proteins.  Plant Phys.. 122 803-812
    Google Scholar
  • 10 DeRisi,  J. L.,, Vishwanath,  R. I.,, and Brown,  P. O.. (1997);  Exploring the metabolic and genetic control of gene expression on a genomic scale.  Science. 278 680-686
    CrossrefPubMedGoogle Scholar
  • 11 Desprez,  T.,, Amselem,  J.,, Caboche,  M.,, and Hofte,  H.. (1998);  Differential gene expression in Arabidopsis monitored using cDNA arrays.  Plant J.. 14 643-652
    CrossrefPubMedGoogle Scholar
  • 12 Elliott,  M. C., and Weston,  G. D.. (1993) Biology and physiology of the sugar-beet plant. The sugar beet crop: science into practice. Cooke, D. A. and Scott, R. K., eds. London; Chapman and Hall pp. 37-66
    Google Scholar
  • 13 Ewing,  B., and Green,  P.. (1998);  Basecalling of automated sequencer traces using Phred. II. Error probabilities.  Genome Res.. 8 186-194
    PubMedGoogle Scholar
  • 14 Fieuw,  S., and Willenbrink,  J.. (1990);  Sugar transport and sugar-metabolizing enzymes in sugar storage roots (Beta vulgaris ssp. altissima).  J. Plant Physiol.. 137 216-223
    PubMedGoogle Scholar
  • 15 Fukuda,  H.. (1997);  Tracheary element differentiation.  The Plant Cell. 9 1147-1156
    PubMedGoogle Scholar
  • 16 Hesse,  H.,, Sonnewald,  U.,, and Willmitzer,  L.. (1995);  Cloning and expression analysis of sucrose-phosphate synthase from sugar beet (Beta vulgaris L.).  Mol. Gen. Genet.. 247 515-520
    PubMedGoogle Scholar
  • 17 Hoheisel,  J. D.,, Maier,  E.,, Mott,  R.,, McCarthy,  L.,, Grigoriev,  A. V.,, Schalkwyk,  L. C.,, Nizetic,  D.,, Francis,  F.,, and Lehrach,  H.. (1993);  High-resolution cosmid and P1 maps spanning the 14-Mbp genome of the fission yeast Schizosaccharomyces pombe. .  Cell. 73 109-120
    CrossrefPubMedGoogle Scholar
  • 18 Kamoun,  S.,, Lindqvist,  H.,, and Govers,  F.. (1997 b);  A novel class of elicitin-like genes from Phytophthora infestans. .  Mol. Plant Microbe Interact.. 10 1028-1030
    PubMedGoogle Scholar
  • 19 Kamoun,  S.,, van West,  P.,, de Jong,  A. J.,, de Groot,  K. E.,, Vleeshouwers,  V. G.,, and Govers,  F.. (1997 a);  A gene encoding a protein elicitor of Phytophthora infestans is down-regulated during infection of potato.  Mol. Plant Microbe Interact.. 10 13-20
    PubMedGoogle Scholar
  • 20 Keegstra,  K., and Raikhel,  N.. (2001);  Plant glycosyltransferases.  Current Opinion in Plant Biology. 4 219-224
    CrossrefPubMedGoogle Scholar
  • 21 Kloos,  D. U.,, Ottmanns,  H.,, Dock,  C.,, Stahl,  D.,, and Hehl,  R.. (2002);  Isolation and molecular analysis of six taproot expressed genes from sugar beet.  Journal of Experimental Botany. 53 1533-1534
    CrossrefPubMedGoogle Scholar
  • 22 Milioni,  D.,, Sado,  P. E.,, Stacey,  N.,, Domingo,  D.,, Roberts,  K.,, and McCann,  M.. (2001);  Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system.  Plant Mol. Biol.. 47 221-238
    CrossrefPubMedGoogle Scholar
  • 23 Miller,  R. T.,, Christoffels,  A. G.,, Gopalakrishnan,  C.,, Burke,  J.,, Ptitsyn,  A. A.,, Broveak,  T. R.,, and Hide,  W. A.. (1999);  A comprehensive approach to clustering of expressed human gene sequence: The Sequence Tag Alignment and Consensus Knowledgebase.  Genome Res.. 9 1143-1155
    CrossrefPubMedGoogle Scholar
  • 24 Ruan,  Y.,, Gilmore,  J.,, and Conner,  T.. (1998);  Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays.  The Plant Journal. 15 821-833
    CrossrefPubMedGoogle Scholar
  • 25 Schlaman,  H. R.,, Risseeuw,  E.,, Franke-van Dijk,  M. E.,, and Hooykaas,  P. J.. (1994);  Nucleotide sequence corrections of the uidA open reading frame encoding beta-glucuronidase.  Gene. 138 259-260
    CrossrefPubMedGoogle Scholar
  • 26 Schneider,  K.,, Borchardt,  D. C.,, Schäfer-Pregl,  R.,, Nagl,  N.,, Glass,  C.,, Jeppsson,  A.,, Gebhardt,  C.,, and Salamini,  F.. (1999);  PCR-based cloning and segregation analysis of functional gene homologues in Beta vulgaris. .  Mol. Gen. Genet.. 262 515-524
    PubMedGoogle Scholar
  • 27 Schneider,  K.,, Schäfer-Pregl,  R.,, Borchardt,  D. C.,, and Salamini,  F.. (2002);  Mapping QTLs for sucrose content, yield and quality in a sugar beet population fingerprinted by EST-related markers.  Theor. Appl. Genet.. 104 1107-1113
    CrossrefPubMedGoogle Scholar
  • 28 Schneider,  K.,, Weisshaar,  B.,, Borchardt,  D. C.,, and Salamini,  F.. (2001);  SNP frequency and allelic haplotype structure of Beta vulgaris expressed genes.  Mol. Breed.. 8 63-74
    CrossrefPubMedGoogle Scholar
  • 29 Scott,  R. K., and Jaggard,  K. W.. (1993) Crop physiology and agronomy. The sugar beet crop: science into practice. Cooke, D. A. and Scott, R. K., eds. London; Chapman and Hall pp. 179-237
    Google Scholar
  • 30 Staden,  R.,, Beal,  K. F.,, and Bonfield,  J. K.. (1998);  The Staden Package.  Methods Mol. Biol. 2000. 132 115-130
    PubMedGoogle Scholar
  • 31 Tesnière,  C., and Verriès,  C.. (2000);  Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development.  Plant Science. 157 77-88
    CrossrefPubMedGoogle Scholar
  • 32 The Arabidopsis Genome Initiative . (2000);  Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. .  Nature. 408 796-815
    CrossrefPubMedGoogle Scholar
  • 33 The Gene Ontology . (2000);  Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.  Nat. Genet.. 25 25-29
    CrossrefPubMedGoogle Scholar
  • 34 Whetten,  R.,, Sun,  Y. H.,, Zhang,  Y.,, Sederoff,  R.. (2001);  Functional genomics and cell wall biosynthesis in loblolly pine.  Plant Mol. Biol.. 47 275-291
    CrossrefPubMedGoogle Scholar
  • 35 Wilkinson,  J. Q.,, Lanahan,  M. B.,, Conner,  T. W.,, and Klee,  H. J.. (1995);  Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display.  Plant Mol. Biol.. 27 1097-1108
    CrossrefPubMedGoogle Scholar
  • 36 Yamada,  S.,, Katsuhara,  M.,, Kelly,  W. B.,, Michalowski,  C. B.,, and Bohnert,  H. J.. (1995);  A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant.  The Plant Cell. 7 1129-1142
    PubMedGoogle Scholar
  • 37 Yu,  J., et al.. (2002);  A draft sequence of the rice genome (Oryza sativa L. ssp. indica).  Science. 296 79-92
    CrossrefPubMedGoogle Scholar
  • 38 Zdobnov,  E. M., and Apweiler,  R.. (2001);  InterProScan - an integration platform for the signature-recognition methods in InterPro.  Bioinformatics. 17 847-848
    CrossrefPubMedGoogle Scholar

K. Schneider

Max-Planck-Institut für Züchtungsforschung

Carl-von-Linné-Weg 10
50829 Köln
Germany

eMail: kschneid@mpiz-koeln.mpg.de

Section Editor: M. Koornneef

      >