Molecular Breeding

, Volume 4, Issue 1, pp 23–31 | Cite as

Rapid and informative assays for Yd2, the barley yellow dwarf virus resistance gene, based on the nucleotide sequence of a closely linked gene

  • C.M. Ford
  • N.G. Paltridge
  • J.P. Rathjen
  • R.L. Moritz
  • R.J. Simpson
  • R.H. Symons


This paper describes the isolation of the cDNA encoding a protein previously shown to be indicative of the disease-resistance phenotype mediated by the Yd2 gene in barley (Hordeum vulgare L.). Amino acid sequences of four peptides obtained after isolation of the protein on two-dimensional polyacrylamide gels were completely homologous to sequences occurring within subunit E of barley vacuolar proton-translocating ATPase. Nucleotide sequence data of cloned cDNAs from both Yd2 and non-Yd2 barley varieties showed an amino acid change arising from a single-base-pair polymorphism. This was predicted to result in the shift in isoelectric point used previously to differentiate the protein in Yd2 and non-Yd2 barleys. Earlier work had indicated very close linkage between the gene from which this cDNA is derived, which we have named Ylp, and Yd2, the barley yellow dwarf virus resistance gene. We report here the development of PCR-based assays which discriminate between the two alleles of Ylp and thereby act as valuable predictors of Yd2 for barley breeders and others looking to study this important gene in cereal crops. The validity of each assay was tested with an extensive survey of over 100 barley varieties currently under cultivation in Australia or of importance to Australian barley breeding programmes. Complete agreement was observed between the allele of Ylp detected by the assay and the known Yd2 status of the barleys. A dominant PCR marker for the Yd2-associated allele of Ylp was subsequently developed using an allele-specific primer pair. This fast and economical assay will have broad application in the marker-assisted selection of Yd2-containing lines.

barley barley yellow dwarf virus diagnostic assay disease resistance gene Yd2 


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  1. 1.
    Altschul SF, Boguski MS, Gish W, Wootton JC: Issues in searching molecular sequence databases. Nature Genet 6: 119–129 (1994).Google Scholar
  2. 2.
    Bassam BJ, Caetano-Anollés G, Gresshoff PM: Fast and sensitive staining of DNA in polyacrylamide gels. Anal Biochem 196: 80–83 (1991).Google Scholar
  3. 3.
    Burnett PA, Comeau A, Qualset CO: Tolerance or resistance for control of barley yellow dwarf. In: D'Arcy CJ, Burnett PA (eds) Barley Yellow Dwarf 40 Years of Progress, pp. 321–343. APS Press, Minnesota (1995).Google Scholar
  4. 4.
    Collins NC: The genetics of barley yellow dwarf virus resistance in barley and rice. Ph.D. thesis, University of Adelaide, Australia (1996).Google Scholar
  5. 5.
    Collins NC, Paltridge NG, Ford CM, Symons RH: The Yd2 gene for barley yellow dwarf virus resistance maps close to the centromere on the long arm of barley chromosome 3. Theor Appl Genet 92: 858–864 (1996).Google Scholar
  6. 6.
    Dietz K-J, Rudloff S, Ageorges A, Eckerskorn C, Fischer K, Arbinger B: Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNAcloning, expression and immunological analysis. Plant J 8: 521–529 (1995).Google Scholar
  7. 7.
    Frohmann MA, Dush MK, Martin GR: Rapid production of full-length cDNA from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002 (1988).Google Scholar
  8. 8.
    Hochstrasser DF, Harrington MG, Hochstrasser A-C, Miller MJ, Merril CR: Methods for increasing the resolution of twodimensional protein electrophoresis. Anal Biochem 173: 424–435 (1988).Google Scholar
  9. 9.
    Holloway PJ, Arundel PH: High-resolution two dimensional electrophoresis of plant proteins. Anal Biochem 172: 8–15 (1988).Google Scholar
  10. 10.
    Holloway PJ, Heath R: Identification of polypeptide markers of barley yellow dwarf virus resistance and susceptibility genes in non-infected barley (Hordeum vulgare) plants. Theor Appl Genet 85: 346–352 (1992).Google Scholar
  11. 11.
    Jakobsen KS, Breivold E, Hornes E: Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres. Nucl Acids Res 18: 3669 (1990).Google Scholar
  12. 12.
    Konieczny A, Ausubel FM: A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCRbased markers. Plant J 4: 403–410 (1993).Google Scholar
  13. 13.
    Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninksy JJ: Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucl Acids Res 18: 999–1005 (1990).Google Scholar
  14. 14.
    Moritz RL, Ward LD, Simpson RJ: High-sensitivity peptide mapping utilizing reversed-phase microbore and microcolumn liquid chromatography. In: Angelletti-Hogue R (ed) Techniques in Protein Chemistry III, pp. 97–106. Academic Press, New York (1992).Google Scholar
  15. 15.
    Moritz RL, Eddes JL, Reid GE, Simpson RJ: Spyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically-separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides. Electrophoresis 17: 907–917 (1996).Google Scholar
  16. 16.
    Program manual for theWisconsin Package, Version 8. Genetics Computer Group, Madison, WI (1994).Google Scholar
  17. 17.
    Puissant C, Houdebaine L-M: An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques 8: 148–149 (1990).Google Scholar
  18. 18.
    Schaller CW, Rasmussen DC, Qualset CO: Sources of resistance to the yellow dwarf virus in barley. Crop Sci 4: 544–548 (1963).Google Scholar
  19. 19.
    Sommer SS, Groszbach AR, Bottema CDK: PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes. Biotechniques 12: 82–87 (1992).Google Scholar
  20. 20.
    Wang H, Qi M, Cutler AJ: A simple method of preparing plant samples for PCR. Nucl Acids Res 21: 4153–4154 (1993).Google Scholar
  21. 21.
    Ward LD, Hong J, Whitehead RH, Simpson RJ: Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 11: 883–891 (1990).Google Scholar
  22. 22.
    Wu DY, Ugozzoli L, Pal BK, Wallace RB: Allele-specific enzymatic amplification of _-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86: 2757–2760 (1989).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • C.M. Ford
    • 1
    • 2
  • N.G. Paltridge
    • 1
  • J.P. Rathjen
    • 1
  • R.L. Moritz
    • 4
  • R.J. Simpson
    • 4
  • R.H. Symons
    • 1
  1. 1.Departments of Plant Science and ofAustralia
  2. 2.Horticulture, Viticulture and OenologyThe University of Adelaide, Waite CampusGlen OsmondAustralia
  3. 3.Center For Engineering Plants for Resistance Against PathogensDavisUSA
  4. 4.Joint Protein Structure LaboratoryLudwig Institute for Cancer Research, The Walter and Eliza Hall Institute of Medical ResearchParkvilleAustralia

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