Polymerase Chain Reaction-Based Markers

  • B. D. Singh
  • A. K. Singh


Restriction fragment length polymorphism (RFLP) was the first DNA-based marker, and it was once widely used in biology and, to some extent, plant breeding. But due to the need for high technical skill, considerable marker development work, and some other limitations, the search continued for more user-friendly DNA marker systems. With the discovery of polymerase chain reaction (PCR) technique, the new generation of PCR-based DNA markers was developed. Initially arbitrary primers of different sizes were used to amplify genomic DNA to generate fingerprints of different individuals. Randomly amplified polymorphic DNAs (RAPDs), DNA amplification fingerprinting (DAF), and arbitrary-primed PCR (AP-PCR) are examples of marker systems based on arbitrary primers. Amplified fragment length polymorphism (AFLP) marker system detects polymorphism due to the sequence variation in and around the recognition sites of restriction endonucleases and uses PCR for marker assay. Refinements in the DNA sequencing technology supported the discovery and development of marker systems, which exploit the sequence variation in specific fragments of DNA using the PCR technology. Sequence-tagged site (STS) markers, including microsatellite or simple sequence repeats (SSR) markers, are an example of this group. The SSR markers revolutionized the marker application in crop improvement in view of their abundance, codominant nature, user-friendliness, and other desirable features. But the development of SSR markers is expensive and complicated so that several other simpler PCR-based marker systems like sequence-related amplification polymorphism (SRAP; it uses open reading frame sequences), target region amplification polymorphism (TRAP; it uses expressed sequence tags), etc. were developed. In addition, some markers exploit variation in RNA sequences of a species: cDNA AFLP, cDNA-SSCP, etc. are examples of RNA-based markers. This chapter describes these and other PCR-based marker systems in some details in addition to introducing the technique of PCR.


Amplify Fragment Length Polymorphism Simple Sequence Repeat Marker Amplify Fragment Length Polymorphism Marker Simple Sequence Repeat Locus Target Region Amplification Polymorphism 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27:617–631PubMedCrossRefGoogle Scholar
  2. Ayliffe MA, Lawrence GJ, Ellis JG (1994) Heteroduplex molecules formed between allelic sequences cause non-parental RAPD bands. Nucleic Acids Res 22:1632–1636PubMedCentralPubMedCrossRefGoogle Scholar
  3. Babu KN, Rajesh MK, Samsudeen K et al (2014) Randomly amplified polymorphic DNA (RAPD) and derived techniques. Methods Mol Biol 1115:191–209PubMedCrossRefGoogle Scholar
  4. Becker J, Heun M (1995) Mapping of digested and undigested random amplified microsatellite polymorphisms in barley. Genome 38:991–998PubMedCrossRefGoogle Scholar
  5. Caetano-Anolles G, Bassam BJ, Greshoff PM (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Bio/Technol 9:553–557CrossRefGoogle Scholar
  6. Caldeira RL, Carvalho OS, Lage RCG et al (2002) Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata. Mem Inst Oswaldo Cruz, Rio de Janeiro 97:23–26CrossRefGoogle Scholar
  7. Chen XM, Line RF, Leung H (1998) Genome scanning for resistance gene analogs in rice, barley and wheat by high-resolution electrophoresis. Theor Appl Genet 97:345–355CrossRefGoogle Scholar
  8. Choi HK, Kim D, Uhm T et al (2004) A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics 166:1463–1502PubMedCentralPubMedCrossRefGoogle Scholar
  9. Choumane W, Winter P, Weigand F et al (2000) Conservation and variability of sequence tagged microsatellite sites (STMSs) from chickpea (Cicer arietinum L.) within the genus Cicer. Theor Appl Genet 101:269–278CrossRefGoogle Scholar
  10. Collard BCY, Mackill DJ (2009a) Start codon targeted (SCoT) polymorphism: a simple, novel DNA marker technique for generating gene-targeted markers in plants. Plant Mol Biol Rep 27:86–93CrossRefGoogle Scholar
  11. Collard BCY, Mackill DJ (2009b) Conserved DNA-derived polymorphism (CDDP): a simple and novel method for generating DNA markers in plants. Plant Mol Biol Rep 27:558–562CrossRefGoogle Scholar
  12. de Vienne D, Santoni S, Falque M (2003) Principal sources of molecular markers. In: de Vienne D (ed) Molecular markers in plant genetics and biotechnology. Science Publishers, Enfield, pp 3–46Google Scholar
  13. Edwards KJ (1998) Randomly amplified polymorphic DNAs (RAPDs). In: Karp A, Isaac PG, Ingram DS (eds) Molecular tools for screening biodiversity. Chapman and Hall, London, pp 171–175CrossRefGoogle Scholar
  14. Ellis THN, Poyser SJ, Knox MR et al (1998) Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea. Mol Gen Genet 260:9–19PubMedGoogle Scholar
  15. Fulton TM, Van der Hoeven R, Eannetta NT et al (2002) Identification, analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants. Plant Cell 14:1457–1467PubMedCentralPubMedCrossRefGoogle Scholar
  16. Gupta PK, Varshney RK (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113:163–185CrossRefGoogle Scholar
  17. Hu J, Vick BA (2003) Target region amplification polymorphism: a novel marker technique for plant genotyping. Plant Mol Biol Rep 21:289–294CrossRefGoogle Scholar
  18. Li G, Quiros CF (2001) 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 103:455–461CrossRefGoogle Scholar
  19. Litt M, Luty JA (1989) A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Human Gene 44:397–401Google Scholar
  20. McGregor CE, Lambert CA, Greyling MM et al (2000) A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica 113:135–144CrossRefGoogle Scholar
  21. Michaels SD, Amasino RM (1998) A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J 14:381–385PubMedCrossRefGoogle Scholar
  22. Mitchelle SE, Kresovich S, Jester CE et al (1997) Application of multiplex PCR and fluorescence-based, semiautomated allele sizing technology for genotyping plant genetic resources. Crop Sci 37:617–624CrossRefGoogle Scholar
  23. Mullis KB (1990) The unusual origin of the polymerase chain reaction. Sci Amer 262:36–43CrossRefGoogle Scholar
  24. Olson ML, Hood L, Cantor C et al (1989) A common language for physical mapping of the human genome. Science 245:1434–1435PubMedCrossRefGoogle Scholar
  25. Orita M, Iwahana H, Kanazawa H et al (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770PubMedCentralPubMedCrossRefGoogle Scholar
  26. Panaud O, Chen X, McCouch SR (1996) Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet 252:597–607PubMedGoogle Scholar
  27. Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85:985–993PubMedCrossRefGoogle Scholar
  28. Poczai P, Varga I, Laos M et al (2013) Advances in plant gene-targeted and functional markers: a review. Plant Methods 9:6. doi: 10.1186/1746-4811-9-6 PubMedCentralPubMedCrossRefGoogle Scholar
  29. Powell W, Morgante M, Andre C et al (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238CrossRefGoogle Scholar
  30. Ribaut J-M, Hu X, Hoisington D et al (1997) Use of STSs and SSRs as rapid and reliable preselection tools is a marker assisted selection - backcross scheme. Plant Mol Biol Rep 15:154–162CrossRefGoogle Scholar
  31. Saiki R, Scharf S, Faloona FA et al (1985) Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354PubMedCrossRefGoogle Scholar
  32. Singh AK, Rana MK, Singh S et al (2014a) CAAT box-derived polymorphism (CBDP): a novel promoter-targeted molecular marker for plants. J Plant Biochem Biotechnol 23:175–183CrossRefGoogle Scholar
  33. Singh VK, Singh AK, Kayastha AM et al (2014b) Bioinformatics for legume genomics research. In: Gupta S, Nadarajan N, Gupta DS (eds) Legumes in the omics era. Springer Science+Business Media, NY, pp 249–275CrossRefGoogle Scholar
  34. Staub JE, Serquen FC, Manju G (1996) Genetic markers, map construction and their application in plant breeding. HortSci 31:729–741Google Scholar
  35. Tautz D (1989) Hypervariability of simple sequences as a general source of polymorphic DNA markers. Nucleic Acids Res 17:6462–6471CrossRefGoogle Scholar
  36. van den Broeck D, Maes T, Sauer M et al (1998) Transposon display identifies individual transposable elements in high copy number lines. Plant J 13:121–129PubMedGoogle Scholar
  37. Vos P, Hogers R, Bleeker R et al (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414PubMedCentralPubMedCrossRefGoogle Scholar
  38. Vosman B (1998) Variations on a theme. In: Karp A, Isaac PG, Ingram DS (eds) Molecular tools for screening biodiversity. Chapman and Hall, London, pp 262–264CrossRefGoogle Scholar
  39. Wang J, Lin M, Crenshaw A et al (2009a) High-throughput single nucleotide polymorphism genotyping using nanofluidic Dynamic Arrays. BMC Genomics 10:561–573PubMedCentralPubMedCrossRefGoogle Scholar
  40. Wang Q, Zhang B, Lu Q (2009b) Conserved region amplification polymorphism (CoRAP) a novel marker technique for plant genotyping in Salivia miltiorrhiza. Plant Mol Biol Rep 27:139–143CrossRefGoogle Scholar
  41. Wang Z, Gerstein M, Snyder M (2009c) RNA-Seq: a revolutionary tool for transcriptomics. Nature Rev Genet 10:57–63PubMedCentralPubMedCrossRefGoogle Scholar
  42. Weber JL, May PE (1989) Abundant class of human DNA polymorphism, which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388–396PubMedCentralPubMedGoogle Scholar
  43. Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213–7218PubMedCentralPubMedCrossRefGoogle Scholar
  44. Williams JGK, Kubelik AR, Livak KJ et al (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:1631–1635CrossRefGoogle Scholar
  45. Williams MNV, Pande N, Nair M et al (1991) Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice (Oryza sativa L.) genomic DNA. Theor Appl Genet 82:489–498PubMedCrossRefGoogle Scholar
  46. Witsenboer H, Vogel J, Michelmore RW (1997) Identification, genetic localization and allelic diversity of amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp). Genome 40:923–926PubMedCrossRefGoogle Scholar
  47. Wu KS, Jones R, Dannaberger L et al (1994) Detection of microsatellite polymorphisms without cloning. Nucleic Acids Res 22:3257–3258PubMedCentralPubMedCrossRefGoogle Scholar
  48. Wu J-M, Li Y-R, Yang L-T et al (2013) cDNA-SCoT: a novel rapid method for analysis of gene differential expression in sugarcane and other plants. Australian J Crop Sci 7:659–664Google Scholar
  49. Zabeau M, Vos P (1993) European patent application. Publication no: EP0534858Google Scholar

Copyright information

© Author(s) 2015

Authors and Affiliations

  • B. D. Singh
    • 1
  • A. K. Singh
    • 2
  1. 1.School of BiotechnologyBanaras Hindu UniversityVaranasiIndia
  2. 2.Division of GeneticsIndian Agricultural Research InstituteNew DelhiIndia

Personalised recommendations