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Theoretical and Applied Genetics

, Volume 85, Issue 8, pp 985–993 | Cite as

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

  • I. Paran
  • R. W. Michelmore
RFLP Report

Summary

Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.

Key words

Molecular marker Disease resistance Lettuce Downy mildew 

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References

  1. 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 252:1651–1656Google Scholar
  2. Farrara B, Ilott TW, Michelmore RW (1987) Genetic analysis of factors for resistance to downy mildew (Bremia lactucae) in lettuce (Lactuca sativa). Plant Pathol 36:499–514Google Scholar
  3. Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SA, Bruns GAP (1990) Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping. Nature 343:774–778Google Scholar
  4. Green ED, Olson MV (1990) Chromosomal region of the cystic fibrosis gene in yeast artifical chromosomes: a model for human genome mapping. Proc Natl Acad Sci USA 87:1213–1217Google Scholar
  5. Horn GT, Richards B, Klinger KW (1989) Amplification of a highly polymorphic VNTR segment by the PCR reaction. Nucleic Acids Res 17:2140Google Scholar
  6. Kesseli RV, Ochoa O, Michelmore RV (1991) Variation at RELP loci in Lactuca spp. and the origin of cultivated lettuce. Genome 34:430–436Google Scholar
  7. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181Google Scholar
  8. Landry BS, Kesseli RV, Farrara B, Michelmore RW (1987) A genetic map of lettuce (Lactuca sativa L.) with restriction fragment length polymorphism, issozyme, disease resistance and morphological markers. Genetics 116:331–337Google Scholar
  9. Maniatis T, Fritsch EF, Sambrook L (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  10. Martin GB, Williams JGK, Tanksley SD (1991) Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogeneic lines. Proc Natl Acad Sci USA 88:2336–2340Google Scholar
  11. Meyers RM, Maniatis T, Lerman LS (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 155:501–527Google Scholar
  12. Meyers RM, Sheffield VC, Cox DR (1988) Detection of single base changes in DNA: ribonuclease clevage and denaturing gradient gel electrophoresis. In: Davies K (ed) Genome analysis: a practical approach. IRL Press, Oxford, pp 95–139Google Scholar
  13. Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations. Proc Natl Acad Sci USA 88:9828–9832Google Scholar
  14. Michelmore RW, Kesseli RV, Francis DM, Paran I, Fortin MG, Yang C-H (1992) Strategies for cloning plant disease resistance genes. In: Gurr S (ed) Molecular plant pathology: a practical approach, vol 2. IRL Press, Oxford, pp 233–287Google Scholar
  15. O'Brien SJ (ed) (1990) Genetic maps. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  16. Olson M, Hood L, Cantor C, Botstein D (1989) A common language for physical mapping of the human genome. Science 245:1434–1435Google Scholar
  17. Paran I, Kesseli RV, Michelmore RW (1991) Identification of RFLP and RAPD markers linked to downy mildew resistance genes in lettuce by using near-isogenic lines. Genome 34:1021–1027Google Scholar
  18. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Coler JL, Kennedy D, Hidaka N, Zsiga M, Buchwald N, Riordan JR, Tsui L, Collins FS (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059–1065Google Scholar
  19. Weber JL (1991) Human DNA polymorphisms based on length-variations in simple sequence tandem repeats. In: Davies KE (ed) Genome analysis vol 1: genetic and physical mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 159–181Google Scholar
  20. Williams JGK, Kubelik AR, Livak KJ, Rafalsky A, Tingey SV (1990) DNA polymorphisms amplified by arbitary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535Google Scholar
  21. Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele specific enzymatic amplification of beta globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86:2757–2760Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • I. Paran
    • 1
  • R. W. Michelmore
    • 1
  1. 1.Department of Vegetable CropsUniversity of CaliforniaDavisUSA

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