Skip to main content
SpringerLink
Log in

Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron

  • Original Paper
  • Published:
Theoretical and Applied Genetics Aims and scope Submit manuscript

Abstract

With a long-term goal of constructing a linkage map of Rhododendron enriched with gene-specific markers, we utilized Rhododendron catawbiense ESTs for the development of high-efficiency (in terms of generating polymorphism frequency) PCR-based markers. Using the gene-sequence alignment between Rhododendron ESTs and the genomic sequences of Arabidopsis homologs, we developed ‘intron-flanking‘ EST–PCR-based primers that would anneal in conserved exon regions and amplify across the more highly diverged introns. These primers resulted in increased efficiency (61% vs. 13%; 4.7-fold) of polymorphism-detection compared with conventional EST–PCR methods, supporting the assumption that intron regions are more diverged than exons. Significantly, this study demonstrates that Arabidopsis genome database can be useful in developing gene-specific PCR-based markers for other non-model plant species for which the EST data are available but genomic sequences are not. The comparative analysis of intron sizes between Rhododendron and Arabidopsis (made possible in this study by aligning of Rhododendron ESTs with Arabidopsis genomic sequences and the sequencing of Rhododendron genomic PCR products) provides the first insight into the gene structure of Rhododendron.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B, Moreno RF, Kerlavage AR, McCombie WR, Venter JC (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252:1651–1656

    Article  PubMed  CAS  Google Scholar 

  • Anderson JA, Churchill GA, Autroque JE, Tanksley SD, Sorrells ME (1993) Optimising parental selection for genetic linkage maps. Genome 36:181–186

    CAS  PubMed  Google Scholar 

  • APG (2003) An update of the Angiosperm Phylogeny Group classification for the orders and families of the flowering plants: APG II. Bot J Linnean Soc 141:399–436

    Google Scholar 

  • Bennett MD, Cox AV, Leitch IJ (1997) Angiosperm DNA c-values database. http://www.rbgkew.org.uk/cval/database1.html

  • Bhalerao R, Keskitalo J, Sterky F, Erlandsson R, Bjorkbacka H, Birve SJ, Karlsson J, Gardestrom P, Gustafsson P, Lundeberg J, Jansson S (2003) Gene expression in autumn leaves. Plant Physiol 131:430–442

    Article  PubMed  Google Scholar 

  • Boguski MS, Schuler GD (1995) Establishing a human transcript map. Nat Genet 10:369–371

    Article  PubMed  CAS  Google Scholar 

  • Cato SA, Gardner RC, Kent J, Richardson TE (2001) A rapid PCR-based method for genetically mapping ESTs. Theor Appl Genet 102:296–306

    Article  CAS  Google Scholar 

  • Choi HK, Kim D, Uhm T, Limpens E, Lim H, Mun J-H, Kalo P, Penmetsa RV, Seres A, Kulikova O, Roe BA, Bisseling T, Kiss GB, Cook DR (2004) A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics 166:1463–1502

    Article  PubMed  CAS  Google Scholar 

  • De Riek J, Dendauw J, Mertens M, De Loose M, Heursel J, Van Bockstaele E (1999) Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders’ collection of evergreen azaleas. Theor Appl Genet 99:1155–1165

    Article  Google Scholar 

  • Deutsch M, Long M (1999) Intron–exon structures of eukaryotic model organisms. Nucleic Acids Res 27:3219–3228

    Article  PubMed  CAS  Google Scholar 

  • Dhanaraj AL, Slovin JP, Rowland LJ (2004) Analysis of gene expression associated with cold acclimation in blueberry floral buds using expressed sequence tags. Plant Sci 166:863–872

    Article  CAS  Google Scholar 

  • Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008

    Article  PubMed  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Dubos C, Plomion C (2003) Identification of water-deficit responsive genes in maritime pine (Pinus pinaster Ait.) roots. Plant Mol Biol 51:249–262

    Article  PubMed  CAS  Google Scholar 

  • Dunemann F, Kahnau R, Stange I (1999) Analysis of complex leaf and flower characters in Rhododendron using a molecular linkage map. Theor Appl Genet 98:1146–1155

    Article  CAS  Google Scholar 

  • Escaravage N, Queestiau S, Pornon A, Doche B, Taberlet P (1998) Clonal diversity in a Rhododendron ferrugineum L. (Ericaceae) population from AFLP markers. Mol Ecol 7:975–982

    Article  CAS  Google Scholar 

  • Eujayl I, Sorrells ME, Baum M, Wolters P, Powell W (2002) Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theor Appl Genet 104:399–407

    Article  PubMed  CAS  Google Scholar 

  • Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14:1675–1690

    Article  PubMed  CAS  Google Scholar 

  • Harry DE, Temesgen B, Neale DB (1998) Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones. Theor Appl Genet 97:327–336

    Article  CAS  Google Scholar 

  • Hudson TJ, Stein LD, Gerety SS, Ma J, Castle AB, Silva J, Slonim DK, Baptista R, Kruglyak L, Xu SH et al (1995) An STS-based map of the human genome. Science 270:1945–1954

    Article  PubMed  CAS  Google Scholar 

  • Iqbal MJ, Paden DW, Rayburn AL (1995) Clonal stability of RAPD markers in three Rhododendron species. J Environ Hort 13:43–46

    CAS  Google Scholar 

  • Komulainen P, Brown GR, Mikkonen M, Karhu A, Garcia-Gil MR, O’Malley D, Lee B, Neale DB, Savolainen O (2003) Comparing EST-based genetic maps between Pinus sylvestris and P. taeda. Theor Appl Genet 107:667–678

    Article  PubMed  CAS  Google Scholar 

  • Leach DG (1961) Rhododendrons of the world and how to grow them. Charles Scribner’s Sons, New York

    Google Scholar 

  • Lim C-C, Krebs SL, Arora R (1999) A 25 kD dehydrin in association with age- and genotype-dependent leaf freezing tolerance in Rhododendron. Theor Appl Genet 99:912–920

    Article  CAS  Google Scholar 

  • Liu ZJ, Corder JF (2004) DNA marker technologies and their applications in aquaculture genetics. Aquaculture 238:1–37

    Article  CAS  Google Scholar 

  • Marian CO, Krebs SL, Arora R (2004) Dehydrin variability among Rhododendron spp: A 25 kDa dehydrin is highly conserved and associated with cold acclimation across a wide array of species. New Phytol 161:773–780

    Article  CAS  Google Scholar 

  • Milbourne D, Meyer R, Bradshow JE, Baird E, Bonar N, Powel JW, Morgante M, Andre C, Hanafey M, Vogel M, Tingey S, Rafalski A (1997) The utility of RFLP, RAPID, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 3:225–238

    Article  Google Scholar 

  • Milla MAR, Butler E, Huete AR, Wilson CF, Anderson O, Gustafson JP (2002) Expressed sequence tag-based gene expression analysis under aluminum stress in rye. Plant Physiol 130:1706–1716

    Article  PubMed  CAS  Google Scholar 

  • Milne RI, Abbott RJ (2000) Origin and evolution of invasive naturalized material of Rhododendron ponticum L. in the British Isles. Mol Ecol 9:541–556

    Article  PubMed  CAS  Google Scholar 

  • Picoult-Newberg L, Ideker TE, Pohl MG, Taylor SL, Donaldson MA, Nickerson DA, Boyce-Jacino M (1999) Mining SNPs from EST databases. Genome Res 9:167–174

    PubMed  CAS  Google Scholar 

  • Plomion C, Hurme P, Frigerio JM, Ridolfi M, Pot D, Pionneau C, Avila C, Gallardo F, David H, Neutelings G, Campbell M, Canovas FM, Savolainen O, Bodenes C, Kremer A (1999) Developing SSCP markers in two Pinus species. Mol Breed 5:21–31

    Article  CAS  Google Scholar 

  • Pornon A, Escaravage N, Thomas P, Taberlet P (2000) Dynamic of genotypic structure in clonal Rhododendron ferrugineum (Ericaceae) population. Mol Ecol 9:1099–1111

    Article  PubMed  CAS  Google Scholar 

  • Rowland LJ, Dhanaraj AL, Polashock JJ, Arora R (2003a) Utility of blueberry-derived EST–PCR primers in related Ericaceae species. HortSci 38:1428–1432

    CAS  Google Scholar 

  • Rowland LJ, Mehra S, Dhanaraj AL, Ogden EL, Slovin JP, Ehlenfeldt MK (2003b) Development of EST–PCR markers for DNA fingerprinting and genetic relationship studies in blueberry (Vaccinium, section Cyanococcus). J Am Soc Hort Sci 128:682–690

    CAS  Google Scholar 

  • Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386

    Google Scholar 

  • Rudd S (2003) Expressed sequence tags: alternative or complement to whole genome sequences? Trends Plant Sci 8:321–329

    Article  PubMed  CAS  Google Scholar 

  • Sakai A, Fuchigami L, Weiser CJ (1986) Cold hardiness in the genus Rhododendron. J Am Soc Hortic Sci 111:273–80

    Google Scholar 

  • Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc Natl Acad Sci USA 93:10614–10619

    Article  PubMed  CAS  Google Scholar 

  • Schlueter SD, Dong Q, Brendel V (2003) GeneSeqer@PlantGDB: gene structure prediction in plant genomes. Nucleic Acids Res 31:3597–3600

    Article  PubMed  CAS  Google Scholar 

  • Senapathy P, Shapiro MB, Harris NL (1990) Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome projects. Methods Enzymol 13:252–278

    Article  Google Scholar 

  • Simpson CG, Leader DJ, Brown JWS (1993) Plant intron sequences. In: Croy RRD (ed) Plant molecular biology labfax. BIOS Scientific Publishers, Oxford, pp 183–251

    Google Scholar 

  • Temesgen B, Brown GR, Harry DE, Kinlaw CS, Sewell MM, Neale DB (2001) Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.). Theor Appl Genet 102:664–675

    Article  CAS  Google Scholar 

  • Thanaraj TA (1999) A clean data set of EST-confirmed splice sites from Homo Sapiens and standards for clean-up procedures. Nucleic Acids Res 27:2627–2637

    Article  PubMed  CAS  Google Scholar 

  • Vainola A (2000) Polyploidization and early screening of Rhododendron hybrids. Euphytica 112:239–244

    Article  Google Scholar 

  • Wei H, Dhanaraj AL, Rowland LJ, Fu Y, Krebs SL, Arora R (2005) Comparative analysis of expressed sequence tags from cold acclimated and non-acclimated leaves of Rhododendron catawbiense Michx. Planta 221:406–416

    Article  PubMed  CAS  Google Scholar 

  • Wright SI, Lauga B, Charlesworth D (2002) Rates and patterns of molecular evolution in inbred and outbred Arabidopsis. Mol Biol Evol 19:1407–1420

    PubMed  CAS  Google Scholar 

  • Wu J, Maehara T, Shimokawa T, Yamamoto S, Harada C, Takazaki Y, Ono N, Mukai Y, Koike K, Yazaki J et al. (2002) A comprehensive rice transcript map containing 6591 expressed sequence tag sites. Plant Cell 14:525–535

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Lisa J. Rowland, Dr. Anik L. Dhanaraj and Wencai Yang for helpful discussions and suggestions.

Author information

Authors and Affiliations

  1. Department of Horticulture, Iowa State University, Ames, IA, 50011, USA

    Hui Wei & Rajeev Arora

  2. Interdepartmental Genetics Graduate Program, Department of Genetics, and Developmental and Cellular Biology, Iowa State University, Ames, IA, 50011, USA

    Yan Fu

Authors
  1. Hui Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yan Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rajeev Arora
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rajeev Arora.

Additional information

Communicated by O. Savolainen

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wei, H., Fu, Y. & Arora, R. Intron-flanking EST–PCR markers: from genetic marker development to gene structure analysis in Rhododendron . Theor Appl Genet 111, 1347–1356 (2005). https://doi.org/10.1007/s00122-005-0064-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00122-005-0064-6

Keywords

Search

Navigation