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.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Andersen JR, Lubberstedt T. Functional markers in plants. Trends Plant Sci. 2003;8:554–50.
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.
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.
Botha AM, Venter E. Molecular marker technology linked to pest and pathogen resistance in wheat breeding. S Afr J Sci. 2000;96:233–40.
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.
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.
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.
Debener T, Mattiesch L. Effective pairwise combination of long primers for RAPD analysis. Plant Breed. 1998;117:147–51.
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.
Farooq S, Azam F. Molecular markers in plant breeding-II. Some prerequisites for use. Pak J Biol Sci. 2002;5:1141–7.
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.
Gostimsky SA, Kokaeva ZG, Konovalov FA. Studying plant genome variation using molecular markers. Russ J Genet. 2005;41:378–88.
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.
Gupta PK, Rustgi S. Molecular markers from the transcribed/expressed region of the genome in higher plants. Funct Integr Geonomics. 2004;4:139–62.
Gupta PK, Varshney RK, Sharma PC, Ramesh B. Molecular markers and their applications in wheat breeding. Plant Breed. 1999;118:369–90.
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.
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.
Hu J, Vick BA. Target region amplification polymorphism: a novel marker technique for plant genotyping. Plant Mol Biol Report. 2003;21:289–94.
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.
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.
Joshi C, Zhou H, Huang X, Chiang VL. Context sequences of translation initiation codon in plants. Plant Mol Biol. 1997;35:993–1001.
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.
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.
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.
Kelly JD, Miklas PN. The role of RAPD markers in breeding for disease resistance in common bean. Mol Breed. 1998;4:1–11.
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.
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.
Mackill DJ. Classifying japonica rice cultivars with RAPD markers. Crop Sci. 1995;35:889–94.
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.
Mueller UG, Wolfenbarger LL. AFLP genotyping and fingerprinting. Trends Ecol Evol. 1999;14:389–94.
Page RDM. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996;12:357–8.
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.
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.
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.
Penner G. RAPD analysis of plant genomes. In: Jauhar PP, editor. Methods of genome analysis in plants. Boca Raton: CRC; 1996. p. 251–268.
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.
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.
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.
Semagn K, Bjornstad A, Ndjiondjop MN. An overview of molecular marker methods for plants. Afr J Biotechnol. 2006;5:2540–68.
Sommer R, Tautz D. Minimal homology requirements for PCR primers. Nucleic Acid Res. 1989;17:6749.
Tanaka J, Taniguchi F. Emphasized-RAPD (e-RAPD): a simple and efficient technique to make RAPD bands clearer. Breed Sci. 2002;52:225–9.
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.
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.
Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990;18:7213–8.
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.
Winter P, Kahl G. Molecular marker technologies for plant improvement. World J Microbiol Biotechnol. 1995;11:438–48.
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.
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.
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.
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, 86–93 (2009). https://doi.org/10.1007/s11105-008-0060-5