Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Abstract

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Andersen JR, Lubberstedt T. Functional markers in plants. Trends Plant Sci. 2003;8:554–50.

    PubMed  Article  CAS  Google Scholar 

  2. Atienzar F, Evenden A, Jha A, Savva D, Depledge M. Optimized RAPD analysis generates high-quality genomic DNA profiles at high annealing temperature. BioTechniques. 2000;28:52–4.

    PubMed  CAS  Google Scholar 

  3. Blair MW, Panaud O, McCouch SR. Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.). Theor Appl Genet. 1999;98:780–92.

    Article  CAS  Google Scholar 

  4. Botha AM, Venter E. Molecular marker technology linked to pest and pathogen resistance in wheat breeding. S Afr J Sci. 2000;96:233–40.

    Google Scholar 

  5. Collard BCY, Das A, Virk PS, Mackill DJ. Evaluation of ‘quick and dirty’ DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.). Plant Breed. 2007;126:47–50.

    Article  CAS  Google Scholar 

  6. Collard BCY, Jahufer MZZ, Brouwer JB, Pang ECK. An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: the basic concepts. Euphytica. 2005;142:169–96.

    Article  CAS  Google Scholar 

  7. Davis TM, Yu H, Haigis KM, McGowan PJ. Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers. Theor Appl Genet. 1995;91:582–8.

    Article  CAS  Google Scholar 

  8. Debener T, Mattiesch L. Effective pairwise combination of long primers for RAPD analysis. Plant Breed. 1998;117:147–51.

    Article  CAS  Google Scholar 

  9. Dziechciarkova M, Lebeda A, Dolezalova I, Astley D. Characterization of Lactuca spp. germplasm by protein and molecular markers—a review. Plant Soil Environ. 2004;50:47–58.

    CAS  Google Scholar 

  10. Farooq S, Azam F. Molecular markers in plant breeding-II. Some prerequisites for use. Pak J Biol Sci. 2002;5:1141–7.

    Article  Google Scholar 

  11. Gillings M, Holley M. Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation. Electrophoresis. 1997;18:1512–8.

    PubMed  Article  CAS  Google Scholar 

  12. Gostimsky SA, Kokaeva ZG, Konovalov FA. Studying plant genome variation using molecular markers. Russ J Genet. 2005;41:378–88.

    Article  CAS  Google Scholar 

  13. Gupta M, Chyi YS, Romero-Severson J, Owen JL. Amplification of DNA markers from evolutionary diverse genomes using single primers of simple-sequence repeats. Theor Appl Genet. 1994;89:998–1006.

    Article  CAS  Google Scholar 

  14. Gupta PK, Rustgi S. Molecular markers from the transcribed/expressed region of the genome in higher plants. Funct Integr Geonomics. 2004;4:139–62.

    CAS  Google Scholar 

  15. Gupta PK, Varshney RK, Sharma PC, Ramesh B. Molecular markers and their applications in wheat breeding. Plant Breed. 1999;118:369–90.

    Article  CAS  Google Scholar 

  16. Hallden C, Hansen M, Nilsson NO, Hjerdin A, Sall T. Competition as a source of errors in RAPD analysis. Theor Appl Genet. 1996;93:185–92.

    Article  Google Scholar 

  17. Holland JB, Helland SJ, Sharopova N, Rhyne DC. Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat. Genome. 2001;44:1065–76.

    PubMed  Article  CAS  Google Scholar 

  18. Hu J, Vick BA. Target region amplification polymorphism: a novel marker technique for plant genotyping. Plant Mol Biol Report. 2003;21:289–94.

    Article  CAS  Google Scholar 

  19. Johnson JR, Clabots C. Improved repetitive-element PCR fingerprinting of Salmonella enterica with the use of extremely elevated annealing temperatures. Clin Diagn Lab Immunol. 2000;7:258–64.

    PubMed  CAS  Google Scholar 

  20. Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F, vandeWiel C, et al. Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Mol Breed. 1997;3:381–90.

    Article  CAS  Google Scholar 

  21. Joshi C, Zhou H, Huang X, Chiang VL. Context sequences of translation initiation codon in plants. Plant Mol Biol. 1997;35:993–1001.

    PubMed  Article  CAS  Google Scholar 

  22. Kalendar R. FastPCR: a PCR primer design and repeat sequence searching software with additional tools for the manipulation and analysis of DNA and protein. Available at www.biocenter.helsinki.fi/bi/programs/fastpcr.htm; 2007.

  23. Kalendar R, Grob T, Regina M, Suoniemi A, Schulman A. IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor Appl Genet. 1999;98:704–11.

    Article  CAS  Google Scholar 

  24. Kaushik A, Saini N, Jain S, Rana P, Singh RK, Jain RK. Genetic analysis of a CSR10 (indica) x Taraori Basmati F3 population segregating for salt tolerance using ISSR markers. Euphytica. 2003;134:231–8.

    Article  CAS  Google Scholar 

  25. Kelly JD, Miklas PN. The role of RAPD markers in breeding for disease resistance in common bean. Mol Breed. 1998;4:1–11.

    Article  CAS  Google Scholar 

  26. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda D, Levenson C, et al. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acid Res. 1990;18:999–1005.

    PubMed  Article  CAS  Google Scholar 

  27. Li G, Quiros CF. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet. 2001;103:455–61.

    Article  CAS  Google Scholar 

  28. Mackill DJ. Classifying japonica rice cultivars with RAPD markers. Crop Sci. 1995;35:889–94.

    CAS  Google Scholar 

  29. Monna L, Miyao A, Inoue T, Fukuoka S, Yamazaki M, Zhong HS, et al. Determination of RAPD markers in rice and their conversion into sequence tagged sites (STSs) and STS-specific primers. DNA Res. 1994;1:138–48.

    Article  Google Scholar 

  30. Mueller UG, Wolfenbarger LL. AFLP genotyping and fingerprinting. Trends Ecol Evol. 1999;14:389–94.

    PubMed  Article  Google Scholar 

  31. Page RDM. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996;12:357–8.

    PubMed  CAS  Google Scholar 

  32. Paran I, Michelmore R. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet. 1993;85:985–93.

    Article  CAS  Google Scholar 

  33. Parsons BJ, Newbury HJ, Jackson MT, Ford-Lloyd BV. Contrasting genetic diversity relationships are revealed in rice (Oryza sativa L.) using different marker types. Mol Breed. 1997;3:115–25.

    Article  CAS  Google Scholar 

  34. Pavlicek A, Hrda S, Flegr J. FreeTree—freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of the genus Frenkelia. Folia Biol (Praha). 1999;45:97–9.

    CAS  Google Scholar 

  35. Penner G. RAPD analysis of plant genomes. In: Jauhar PP, editor. Methods of genome analysis in plants. Boca Raton: CRC; 1996. p. 251–268.

    Google Scholar 

  36. Perry AL, Worthington T, Hilton AC, Lambert PA, Stirling AJ, Elliott TSJ. Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD. FEMS Microbiol Lett. 2003;228:51–5.

    PubMed  Article  CAS  Google Scholar 

  37. Rao KK, Lakshminarasu M, Jena KK. DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice. Biotechnol Adv. 2002;20:33–47.

    PubMed  Article  CAS  Google Scholar 

  38. Sawant SV, Singh PK, Gupta SK, Madnala R, Tuli R. Conserved nucleotide sequences in highly expressed genes in plants. J Genet. 1999;78:123–31.

    Article  CAS  Google Scholar 

  39. Semagn K, Bjornstad A, Ndjiondjop MN. An overview of molecular marker methods for plants. Afr J Biotechnol. 2006;5:2540–68.

    CAS  Google Scholar 

  40. Sommer R, Tautz D. Minimal homology requirements for PCR primers. Nucleic Acid Res. 1989;17:6749.

    PubMed  Article  CAS  Google Scholar 

  41. Tanaka J, Taniguchi F. Emphasized-RAPD (e-RAPD): a simple and efficient technique to make RAPD bands clearer. Breed Sci. 2002;52:225–9.

    Article  CAS  Google Scholar 

  42. Tyler KD, Wang G, Tyler SD, Johnson WM. Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J Clin Microbiol. 1997;35:339–46.

    PubMed  CAS  Google Scholar 

  43. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hoernes M, et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995;23:4407–14.

    PubMed  Article  CAS  Google Scholar 

  44. Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990;18:7213–8.

    PubMed  Article  CAS  Google Scholar 

  45. Williams J, Kubelik A, Livak K, Rafalski J, Tingey S. DNA Polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990;18:6531–5.

    PubMed  Article  CAS  Google Scholar 

  46. Winter P, Kahl G. Molecular marker technologies for plant improvement. World J Microbiol Biotechnol. 1995;11:438–48.

    Article  CAS  Google Scholar 

  47. Ye G-N, Hemmat M, Lodhi MA, Weeden NF, Reisch BI. Long primers for RAPD mapping and fingerprinting of grape and pear. BioTechniques. 1996;20:368–71.

    PubMed  CAS  Google Scholar 

  48. Zheng K, Subudhi PK, Domingo J, Maopantay G, Huang N. Rapid DNA isolation for marker assisted selection in rice breeding. Rice Genet Newsl. 1995;12:48.

    Google Scholar 

Download references

Acknowledgements

Technical assistance for gel electrophoresis by Ms. Miladie Penarubia is gratefully acknowledged. We also thank Dr. C. Raghavan and two anonymous reviewers for valuable comments on the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Bertrand C. Y. Collard.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Collard, B.C.Y., Mackill, D.J. Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants. Plant Mol Biol Rep 27, 86 (2009). https://doi.org/10.1007/s11105-008-0060-5

Download citation

Keywords

  • Gene-targeted markers
  • Start codon
  • Genetic diversity
  • QTL mapping