Molecular Breeding

, Volume 2, Issue 3, pp 225–238 | Cite as

The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

  • Wayne Powell
  • Michele Morgante
  • Chaz Andre
  • Michael Hanafey
  • Julie Vogel
  • Scott Tingey
  • Antoni Rafalski
Research Paper


The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.

Key words

AFLP SSR simple sequence repeat polymorphism germplasm microsatellite polymorphism RAPD RFLP soybean Glycine 



restriction fragment length plymorphism


random-amplified polymorphic DNA


amplified fragment length polymorphism


simple sequence repeat


polymerase chain reaction


Tris-borate-EDTA buffer


marker index


sum of effective numbers of alleles


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  1. 1.
    Akkaya MS, Bhagwat AA, Cregan PB: Length polymorphism of simple sequence repeat DNA in soybean. Genetics 132: 1131–1139 (1992).Google Scholar
  2. 2.
    Beckmann JS, Soller M: Restriction fragment length polymorphisms in genetic improvement: methodologies, mapping and costs. Theor Appl Genet 67: 35–43 (1983).Google Scholar
  3. 3.
    Bell CJ, Ecker JR: Assignment of thirty microsatellite loci to the linkage map ofArabidopsis. Genomics 19: 137–144 (1994).Google Scholar
  4. 4.
    Botstein D, White RL, Skolnick MH, Davis RW: Construction of a genetic map in man using restriction fragment length polymorphisms. Am J Hum Genet 32: 314–331 (1980).Google Scholar
  5. 5.
    Bowcock AM, Ruiz-Linares A, Tomfohrde J, Minch E, Kidd JR, Cavalli-Sforza LL: High resolution of human evolutionary trees with polymorphic microsatellites. Nature 368: 455–457 (1994).Google Scholar
  6. 6.
    Caetano-Anolles G, Bassam BJ, Gresshoff PM: High resolution DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Bio/technology 9: 553–557 (1991).Google Scholar
  7. 7.
    Coe JE.H., Neuffer MG, Hoisington DA: The genetics of corn. In: Sprague GF, Dudley JW (eds) Corn and Corn Improvement, pp. 81–258. American Society of Agronomy, Madison, WI (1988).Google Scholar
  8. 8.
    Cornall RJ, Aitman TJ, Hearne CM, Todd JA: The generation of a library of PCR-analyzed microsatellite variants for genetic mapping of the mouse genome. Genomics 10: 874–881 (1991).Google Scholar
  9. 9.
    Delanney X, Rodgers DM, Palmer RG: Relative genetic contributions among ancestral lines to North-American soybean cultivars. Crop Sci 23: 944–949 (1983).Google Scholar
  10. 10.
    Dietrich W, Katz H, Lincoln SE, Shin H-S, Friedman J, Dracopoli NC, Lander ES: A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131: 423–447 (1992).Google Scholar
  11. 11.
    dosSantos JB, Nienhuis J, Skroch P, Tivang J, Slocum MK: Comparison of RAPD and RFLP genetic markers in determining genetic similarity amongBrassica oleracea L. genotypes. Theor Appl Genet 87: 909–915 (1994).Google Scholar
  12. 12.
    Gregorius HR: The relationship between the concepts of genetic diversity and differentiation. Theor Appl Genet 74: 397–401 (1987).Google Scholar
  13. 13.
    Hallden C, Nilsson N-O, Rading IM, Saell T: Evaluation of RFLP and RAPD markers in comparison of Brassica napus breeding lines. Theor Appl Genet 88: 123–128 (1994).Google Scholar
  14. 14.
    Hymowitz T, Bernard RL: Origin of the soybean and germplasm introduction and development in North American. In:Use of Plant Introductions in Cultivar Development, Part I CSSA Special Publication 17, pp. 147–164. Crop Science Society of America, Madison, WI (1991).Google Scholar
  15. 15.
    Lawler FC, Stoffel S, Saiki RK, Chang S-Y, Landre PA, Abramson RD, Gelfand DH: High-level expression, purification, and enzymatic characterization of full-lengthThermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. PCR Meth Appl 2: 275–287 (1993).Google Scholar
  16. 16.
    Li Z, Furnier GR: Comparison of allozyme, RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen. Theor Appl Genet 87: 97–105 (1993).Google Scholar
  17. 17.
    Lin J-J, Kuo J: AFLPTM: A Novel PCR-Based Assay for Plant and Bacterial DNA Fingerprinting. Focus 17: 66–70 (1995).Google Scholar
  18. 18.
    Mantel N: The dedection of disease clustering and a generalized regression approach. Cancer Res 27: 209–220 (1967).Google Scholar
  19. 19.
    Marshall DR, Allard RW: Isozyme polymorphisms in natural populations ofAven fatua andAvena barbata. Heredity 25: 373–382 (1970).Google Scholar
  20. 20.
    Morgante M, Olivieri AM: PCR-amplified microsatellites as markers in plant genetics. Plant J 3: 175–182 (1993).Google Scholar
  21. 21.
    Morgante M, Rafalski JA, Biddle P, Tingey S, Olivieri AM: Genetic mapping and variability of seven soybean simple sequence repeat loci. Genome 37: 763–769 (1994).Google Scholar
  22. 22.
    Nei M: Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA 70: 3321–3323 (1973).Google Scholar
  23. 23.
    Nei M, Li WH: Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76: 5269–5273 (1979).Google Scholar
  24. 24.
    Parks C, Chang L-S, Shenk T: A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region. Nucl Acids Res 19: 7155–7160 (1991).Google Scholar
  25. 25.
    Peakall R, Smouse PE, Huff DR: Evolutionary Implications of allozyme and RAPD variation in diploid populations of dioecious buffalograssBuchloe dactyloides. Mol Ecol 4: 135–147 (1995).Google Scholar
  26. 26.
    Rafalski A, Tingey S: RFLP map of soybean (Glycine max) 2n=40. In: O'Brien SJ (ed) Genetic Maps. Locus Maps of Complex Genomes. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1993).Google Scholar
  27. 27.
    Rafalski A, Tingey S, Williams JGK: Random amplified polymorphic DNA (RAPD) markers. In: Gelvin SB, Schilperoort RA, Eds. Plant Molecular Biology Manual, pp. 1–8. Kluwer Academic Publishers, Dordrecht (1994).Google Scholar
  28. 28.
    Rafalski JA, Morgante M, Powell W, Vogel JM, Tingey SV: Generating and using DNA markers in plants. In: Birren B, Lai E, (eds)Analysis of Non-mammalian Genomes: A practical Guide, press. Academic Press, Boca Raton, FL (1996).Google Scholar
  29. 29.
    Rafalski JA, Tingey SV: Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines. Trends Genet 9: 275–280 (1993).Google Scholar
  30. 30.
    Rohlf FJ: NTSYS-pc Numerical Taxonomy and Multivariate Analysis System version 1.7. Owner manual (1992).Google Scholar
  31. 31.
    Rongwen J, Akkaya MS, Bhagwat AA, Lavi U, Cregan PB: The use of microsatellite DNA markers for soybean genotype identification. Theor Appl Genet 90: 43–48 (1995).Google Scholar
  32. 32.
    Rus-Kortekass W, Smulders MJM, Arens P, Vosman B: Direct comparison of levels of genetic variation in tomato detected by a GACA-containing microsatellite probe and by random amplified polymorphic DNA. Genome 37: 375–381 (1994).Google Scholar
  33. 33.
    Saghai Maroof MA, Biyashev RM, Yang GP, Zhang Q, Allard RW: Extraordinarily polymorphic microsatellite DNA in Barley: Species diversity, chromosomal locations, and population dynamics. Proc Natl Acad Sci USA 91: 5466–5470 (1994).Google Scholar
  34. 34.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).Google Scholar
  35. 35.
    Senior ML, Heun M: Mapping maize microsatellites and polymerase chain reaction confirmation of the targeted repeats using a CT primer. Genome 36: 884–889 (1993).Google Scholar
  36. 36.
    Serikawa T, Kuramoto T, Hilbert P, Mori M, Yamada J, Dubay CJ, Lindpainter K, Ganten D, Guenet J-L, Lathrop GM, Beckman JS: Rat gene mapping using PCR-analyzed microsatellites. Genetics 131: 701–721 (1992).Google Scholar
  37. 37.
    Smith OS, Smith JSC, Bowen SL, Tenborg RA, Wall SJ: Similarities among a group of elite maize inbreds as measured by pedigree, F1 grain-yield, heterosis and RFLPs. Theor Appl Genet 80: 833–840 (1990).Google Scholar
  38. 38.
    Sokal RR, Michener CD: A statistical method for evaluating systematic relationships. Univ Kansas Sci Bull 38: 1409–1438 (1958).Google Scholar
  39. 39.
    Tautz D: Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucl Acids Res 17: 6463–6471 (1989).Google Scholar
  40. 40.
    Tautz D, Renz M: Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucl Acids Res 12: 4127–4138 (1984).Google Scholar
  41. 41.
    Tautz D, Trick M, Dover GA: Cryptic simplicity in DNA is a major source of genetic variation. Nature 322: 652–656 (1986).Google Scholar
  42. 42.
    Thomas MR, Cain P, Scott NS: DNA typing of grapwvine: A universal methodology and database for describing cultivars and evaluating genetic relatedness. Plant Mol Biol 25: 939–949 (1994).Google Scholar
  43. 43.
    Thomas MR, Scott NS: Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theor Appl Genet 86: 985–990 (1993).Google Scholar
  44. 44.
    Thormann CE, Ferreira ME, Camargo LEA, Tivang JG, Osborn TC: Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species. Theor Appl Genet 88: 973–980 (1994).Google Scholar
  45. 45.
    Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarecky D, Ayala FJ: Genetic characterization of six parazitic protozoa: Parity between random-primer DNA typing and multilocus enzyme electrophoresis. Proc Natl Acad Sci USA 90: 1335–1339 (1993).Google Scholar
  46. 46.
    Tingey SV, delTufo JP: Genetic Analysis with RAPD Marklers. Plant Physiol 101: 349–352 (1993).Google Scholar
  47. 47.
    Todd JA: La carte des microsatellites est arrivé! Human Mol Genet 1: 663–666 (1992).Google Scholar
  48. 48.
    Waugh R, Powell W: Using RAPD markers for crop improvement. Trends Bio/technol 10: 186–191 (1992).Google Scholar
  49. 49.
    Weber J, May PE: Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44: 388–396 (1989).Google Scholar
  50. 50.
    Weissenbach J Ed.: The Genethon microsatellite map catalogue. Genethon, Evry, France (1992).Google Scholar
  51. 51.
    Welsh J, McClelland M: Fingerprinting genomes using PCR with arbitrary primers. Nucl Acids Res 18: 7213–7218 (1990).Google Scholar
  52. 52.
    Whitkus R, Doebley J, Wendel JF: Nuclear DNA Markers in systematics and evolution. In: Phillips RL, Vasil IK (eds) DNA Based Markers in Plants pp. 116–141. Kluwer Academic Publishers, Dordrecht (1994).Google Scholar
  53. 53.
    Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res 18: 6531–6535 (1990).Google Scholar
  54. 54.
    Williams JGK, Rafalski JA, Tingey SV: Genetic analysis using RAPD markers. Meth Enzymol 218: 704–780 (1993).Google Scholar
  55. 55.
    Wu K-S, Tanksley SD: Abundance, polymorphism and genetic mapping of microsatellites in rice. Mol Gen Genet 241: 225–235 (1993).Google Scholar
  56. 56.
    Yang GP, Saghai Maroof MA, Xu CG, Zhang Q, Biyashev RM: Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice. Mol Gen Genet 245: 187–194 (1994).Google Scholar
  57. 57.
    Zabeau M, Voss P: Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Application 92402629.7 (1993).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Wayne Powell
    • 1
  • Michele Morgante
    • 1
  • Chaz Andre
    • 1
  • Michael Hanafey
    • 1
  • Julie Vogel
    • 1
  • Scott Tingey
    • 1
  • Antoni Rafalski
    • 1
  1. 1.Biotechnology Research, Agricultural Products DepartmentE.I. du Pont de Nemours and Co (Inc.)WilmingtonUSA

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