Theoretical and Applied Genetics

, Volume 112, Issue 2, pp 358–365 | Cite as

Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences

  • P. C. Bundock
  • M. J. Cross
  • F. M. Shapter
  • R. J. Henry
Original Paper

Abstract

Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.

Notes

Acknowledgements

The authors would like to thank Dr Agnelo Furtado and Mr Daniel Waters for comments on the manuscript. This work was funded by the Molecular Plant Breeding Co-operative Research Centre.

Supplementary material

122_2005_137_MOESM1_ESM.pdf (71 kb)
Supplementary material

References

  1. Bundock PC, Christopher JT, Eggler P, Ablett G, Henry RJ, Holton TA (2003) Single nucleotide polymorphisms in cytochrome P450 genes from barley. Theor Appl Genetics 106:676–682Google Scholar
  2. Chiapparino E, Lee D, Donini P (2004) Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR. Genome 47:414–420PubMedCrossRefGoogle Scholar
  3. Graner A, Kota R, Perovic D, Potokina E, Prasad M, Scholz U, Stein N, Thiel T, Varshney RK, Zhang H (2004) Molecular mapping: shifting from the structural to the functional level. In: Oral presentations. Proceedings 9th International Barley Genetics Symposium, Brno, Czech Republic, 20–26 June 2004, p 49Google Scholar
  4. Gupta PK, Roy JK, Prasad M (2001) Single nucleotide polymorphisms: a new paradigm for molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Sci 80:524–535Google Scholar
  5. Gut IG (2001) Automation in genotyping of single nucleotide polymorphisms. Hum Mutat 17:475–492CrossRefPubMedGoogle Scholar
  6. Hamajima N, Saito T, Matsuo K, Kozaki K, Takahashi T, Tajima K (2000) Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Jpn J Cancer Res 91:865–868PubMedGoogle Scholar
  7. Holton TA, Christopher JT, McClure L, Harker N, Henry RJ (2002) Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat. Mol Breeding 9:63–71CrossRefGoogle Scholar
  8. Imyanitov EN, Buslov KG, Suspitsin EN, Kuligina ES, Belogubova EV, Grigoriev MY, Togo AV, Hanson KP (2002) Improved reliability of allele-specific PCR. Biotechniques 33:484–490PubMedGoogle Scholar
  9. Kanazin V, Talbert H, See D, DeCamp P, Nevo E, Blake T (2002) Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare). Plant Mol Biol 48:529–537CrossRefPubMedGoogle Scholar
  10. Kota R, Varshney RK, Thiel T, Dehmer KJ, Graner A (2001) Generation and comparison of EST-derived SSRs and SNPs in barley (Hordeum vulgare L.). Hereditas 135:145–151CrossRefPubMedGoogle Scholar
  11. Kota R, Rudd S, Facius A, Kolesov G, Thiel T, Zhang H, Stein N, Mayer K, Graner A (2003) Snipping polymorphisms from large EST collections in barley (Hordeum vulgare L.). Mol Gen Genomics 270:24–33CrossRefGoogle Scholar
  12. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18:999–1005PubMedCrossRefGoogle Scholar
  13. Liu Q, Thorland EC, Heit JA, Sommer SS (1997) Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes. Genome Res 7:389–398PubMedGoogle Scholar
  14. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503–2516PubMedCrossRefGoogle Scholar
  15. Rudd S, Mewes H-W, Mayer KFX (2003) Sputnik: a database platform for comparative plant genomics. Nucleic Acids Res 31:128–132 CrossRefPubMedGoogle Scholar
  16. Sachidanandam R, Weissman D, Schmidt SC, Kakol JM, Stein LD, Mullikin JC, Mortimore BJ, Willey DL, Hunt SE, Cole CG et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409:928–933PubMedCrossRefGoogle Scholar
  17. Sato K, Nankaku N, Motoi Y and Takeda K (2004) A large scale mapping of ESTs on the barley genome. In: Oral presentations. Proceedings 9th International Barley Genetics Symposium, Brno, Czech Republic, 20–26 June 2004, p 79 Google Scholar
  18. Schork NJ, Fallin D, Lanchbury JS (2000) Single nucleotide polymorphisms and the future of genetic epidemiology. Clin Genetics 58:250–264CrossRefGoogle Scholar
  19. Soleimani VD, Baum BR, Johnson DA (2003) Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR, with an example from barley. Plant Mol Biol Rep 21:281–288CrossRefGoogle Scholar
  20. Varshney RK, Thiel T, Stein N, Langridge P, Graner A (2002) In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cell Mol Biol Lett 7:537–546PubMedGoogle Scholar
  21. Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA 86:2757–2760PubMedCrossRefGoogle Scholar
  22. Ye S, Dhillon S, Ke XY, Collins AR, Day INM (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:e88CrossRefPubMedGoogle Scholar
  23. Ye S, Humphries S, Green F (1992) Allele specific amplification by tetra-primer PCR. Nucleic Acids Res 20:1152PubMedCrossRefGoogle Scholar
  24. Zhang W, Gianibelli MC, Ma W, Rampling L, Gale KR (2003) Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum. Theor Appl Genet 107:130–138CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • P. C. Bundock
    • 1
  • M. J. Cross
    • 1
  • F. M. Shapter
    • 1
  • R. J. Henry
    • 1
  1. 1.Centre for Plant Conservation Genetics, Molecular Plant Breeding CRCSouthern Cross UniversityLismoreAustralia

Personalised recommendations