Theoretical and Applied Genetics

, Volume 91, Issue 4, pp 691–698

A molecular method for S-allele identification in apple based on allele-specific PCR

  • G. A. Janssens
  • I. J. Goderis
  • W. F. Broekaert
  • W. Broothaerts


cDNA sequences corresponding to two self-incompatibility alleles (S-alleles) of the apple cv ‘Golden Delicious’ have previously been described, and now we report the identification of three additional S-allele cDNAs of apple, one of which was isolated from a pistil cDNA library of cv ‘Idared’ and two of which were obtained by reverse transcription-PCR (RT-PCR) on pistil RNA of cv ‘Queen's Cox’. A comparison of the deduced amino acid sequences of these five S-allele cDNAs revealed an average homology of 69%. Based on the nucleotide sequences of these S-allele cDNAs, we developed a molecular technique for the diagnostic identification of the five different S-alleles in apple cultivars. The method used consists of allele-specific PCR amplification of genomic DNA followed by digestion of the amplification product with an allele-specific restriction endonuclease. Analysis of a number of apple cultivars with known S-phenotype consistently showed coincidence of phenotypic and direct molecular data of the S-allele constitution of the cultivars. It is concluded that the S-allele identification approach reported here provides a rapid and useful method to determine the S-genotype of apple cultivars.

Key words

Malus x domestica Apple Self-incompatibility S-alleles PCR 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aldrich J, Cullis CA (1993) RAPD analysis in flax: optimisation of yield and reproduction using Klen TaqI DNA polymerase, Chelex 100, and gel purification of genomic DNA. Plant Mol Biol Rep 11:128–141Google Scholar
  2. Brace J, King GJ, Ockendon DJ (1994) A molecular approach to the identification of S-alleles in Brassica oleracea. Sex Plant Reprod 7:203–208Google Scholar
  3. Broothaerts W, Vanvinckenroye P, Decock B, Van Damme J, Vendrig JC (1991) Petunia hybrida S-proteins:ribonuclease acitivity and the role of their glycan side chains in self-incompatibility. Sex Plant Reprod 4:258–266Google Scholar
  4. Broothaerts W, Janssens GA, Proost P, Broekaert WF (1995) cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. Plant Mol Biol 27, 499–511Google Scholar
  5. Clarke AE, Newbegin ED (1993) Molecular aspects of self-incompatibility in flowering plants. Annu Rev Genet 27:257–279Google Scholar
  6. Dayhoff MO, Schwarts RM, Orcutt BC (1979) A model of evolutionary change in proteins. In: Dayhoff MO (ed) Atlas of protein sequence and structure, vol 5 (suppl 3). National Biomedical Research Foundation, Washington D.C., pp. 345–352Google Scholar
  7. de Nettancourt D (1977) Incompatibility in angiosperms. In: Frankel R, Gall GAE, Linskens HF (eds) Monographs in theoretical and applied genetics, vol 3. Springer, Berlin Heidelberg New YorkGoogle Scholar
  8. Green PJ (1994) The ribonucleases of higher plants. Annu Rev Plant Physiol Plant Mol Biol 45:421–445Google Scholar
  9. Ioerger TR, Clark AG, Kao T-h (1990) Polymorphism at the self-incompatibility locus in Solanaceae predates speciation. Proc Natl Acad Sci USA 87:9732–9735Google Scholar
  10. Ioerger TR, Gohlke JR, Xu B, Kao T-h (1991) Primary structural features of the self-incompatibility protein in Solanaceae. Sex Plant Reprod 4:81–87Google Scholar
  11. Kaufmann H, Salamini F, Thompson RD (1991) Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum. Mol Gen Genet 226:457–466Google Scholar
  12. Kawata Y, Sakiyama F, Tamaoki H (1988) Amino acid sequence of ribonuclease T2 from Aspergillus oryzae. Eur J Biochem 176:683–697Google Scholar
  13. Kawata Y, Sakiyama F, Hayashi F, Kyogoku Y (1990) Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae. Eur J Biochem 187:255–262Google Scholar
  14. Kobel F, Steinegger P, Anliker J (1939) Weitere Untersuchungen über die Befruchtungsverhältnisse der Apfel-und Birnensorten. Landw Jb Schweiz pp 160–191Google Scholar
  15. Lee H-s, Singh A, Kao T-h (1992) RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequence similarity with solanaceous S-proteins. Plant Mol Biol 20:1131–1141Google Scholar
  16. Lee H-s, Huang S, Kao T-h (1994) S-protein rejection of incompatible pollen in Petunia inflata. Nature 367:560–563Google Scholar
  17. Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20PubMedGoogle Scholar
  18. Manganaris AG, Alston FH (1987) Inheritance and linkage relationships of glutamate oxaloacetate transaminae isoenzymes in apple. I. The GOT-1, a marker for the S-incompatibility locus. Theor Appl Genet 74:154–161Google Scholar
  19. McClure BA, Haring V, Ebert PR, Anderson MA, Simpson RJ, Sakiyama F, Clarke AE (1989) Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature 342:955–957CrossRefPubMedGoogle Scholar
  20. Murfett J, Atherton TL, Mou B, Gasser CS, McClure BA (1994) S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection. Nature 367:563–566Google Scholar
  21. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  22. Sassa H, Hirano H, Ikehashi H (1992) Self-incompatibility-related RNases in styles of Japanese pear (Pyrus serotina Redh.) Plant Cell Physiol 33:811–814Google Scholar
  23. Sassa H, Hirano H, Ikehashi H (1993) Identification and characterization of stylar glycoproteins associated with self-incompatibility genes of Japanese pear, Pyrus serotina Redh. Mol Gen Genet 241:17–25Google Scholar
  24. Sassa H, Mase N, Hirano H, Ikehashi H (1994) Identification of selfincompatibility-related glycoproteins in styles of apple (Malus x domestica). Theor Appl Genet 89:201–205Google Scholar
  25. Singh A, Ai Y, Kao T-h (1991) Characterization of ribonuclease activity of three S-allele-associated proteins of Petunia inflata. Plant Physiol 96:61–68Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • G. A. Janssens
    • 1
    • 2
  • I. J. Goderis
    • 1
  • W. F. Broekaert
    • 1
  • W. Broothaerts
    • 2
  1. 1.F. A. Janssens Laboratory of Genetics, Katholieke Universiteit LeuvenLeuvenBelgium
  2. 2.Centre for Fruit Culture, Katholieke Universiteit LeuvenLeuvenBelgium

Personalised recommendations