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IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Abstract

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1–2 d.

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Figure 1: Retrotransposon-based marker methods.
Figure 2: IRAP gel fingerprints.
Figure 3: IRAP gel fingerprints.

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References

  1. Feschotte, C., Jiang, N. & Wessler, S. Plant transposable elements: where genetics meets genomics. Nat. Rev. Genet. 3, 329–341 (2002).

    Article  CAS  Google Scholar 

  2. Sabot, F. & Schulman, A.H. Parasitism and the retrotransposon life cycle in plants: a hitchhiker's guide to the genome. Heredity 97, 381–388 (2006).

    Article  CAS  Google Scholar 

  3. Schulman, A.H. & Kalendar, R. A movable feast: diverse retrotransposons and their contribution to barley genome dynamics. Cytogenet. Genome Res. 110, 598–605 (2005).

    Article  CAS  Google Scholar 

  4. Hedges, D.J. & Batzer, M.A. From the margins of the genome: mobile elements shape primate evolution. Bioessays 27, 785–794 (2005).

    Article  CAS  Google Scholar 

  5. Ostertag, E.M. & Kazazian, H.H. Genetics: LINEs in mind. Nature 435, 890–891 (2005).

    Article  CAS  Google Scholar 

  6. Jurka, J. Evolutionary impact of human Alu repetitive elements. Curr. Opin. Genet. Dev. 14, 603–608 (2004).

    Article  CAS  Google Scholar 

  7. Waugh, R. et al. Genetic distribution of BARE-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Gen. Genet. 253, 687–694 (1997).

    Article  CAS  Google Scholar 

  8. Leigh, F. et al. Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques. Mol. Genet. Genomics 269, 464–474 (2003).

    Article  CAS  Google Scholar 

  9. Queen, R.A., Gribbon, B.M., James, C., Jack, P. & Flavell, A.J. Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat. Mol. Genet. Genomics 271, 91–97 (2004).

    Article  CAS  Google Scholar 

  10. Nagy, E.D., Molnar, I., Schneider, A., Kovacs, G. & Molnar-Lang, M. Characterization of chromosome-specific S-SAP markers and their use in studying genetic diversity in Aegilops species. Genome 49, 289–296 (2006).

    Article  CAS  Google Scholar 

  11. Yu, G.-X. & Wise, R.P. An anchored AFLP- and retrotransposon-based map of diploid Avena. Genome 43, 736–749 (2000).

    Article  CAS  Google Scholar 

  12. Venturi, S., Dondini, L., Donini, P. & Sansavini, S. Retrotransposon characterisation and fingerprinting of apple clones by S-SAP markers. Theor. Appl. Genet. 112, 440–444 (2006).

    Article  CAS  Google Scholar 

  13. Lanteri, S. et al. A first linkage map of globe artichoke (Cynara cardunculus var. scolymus L.) based on AFLP, S-SAP, M-AFLP and microsatellite markers. Theor. Appl. Genet. 112, 1532–1542 (2006).

    Article  CAS  Google Scholar 

  14. Syed, N.H. et al. A detailed linkage map of lettuce based on SSAP, AFLP and NBS markers. Theor. Appl. Genet. 112, 517–527 (2006).

    Article  CAS  Google Scholar 

  15. Jing, R. et al. Insertional polymorphism and antiquity of PDR1 retrotransposon insertions in Pisum species. Genetics 171, 741–752 (2005).

    Article  CAS  Google Scholar 

  16. Porceddu, A. et al. Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L. Mol. Genet. Genomics 267, 107–114 (2002).

    Article  CAS  Google Scholar 

  17. Ellis, T.H.N., Poyser, S.J., Knox, M.R., Vershinin, A.V. & Ambrose, M.J. Ty1-copia class retrotransposon insertion site polymorphism for linkage and diversity analysis in pea. Mol. Gen. Genet. 260, 9–19 (1998).

    CAS  PubMed  Google Scholar 

  18. Tam, S.M. et al. Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR. Theor. Appl. Genet. 110, 819–831 (2005).

    Article  CAS  Google Scholar 

  19. Tahara, M. et al. Isolation of an active element from a high-copy-number family of retrotransposons in the sweetpotato genome. Mol. Genet. Genomics 272, 116–127 (2004).

    Article  CAS  Google Scholar 

  20. Vicient, C.M. et al. Retrotransposon BARE-1 and its role in genome evolution in the genus Hordeum. Plant Cell 11, 1769–1784 (1999).

    Article  CAS  Google Scholar 

  21. Ramsay, L. et al. Intimate association of microsatellite repeats with retrotransposons and other dispersed repetitive elements in barley. Plant J. 17, 415–425 (1999).

    Article  CAS  Google Scholar 

  22. Zietkiewicz, E., Rafalski, A. & Labuda, D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20, 176–183 (1994).

    Article  CAS  Google Scholar 

  23. Flavell, A.J., Knox, M.R., Pearce, S.R. & Ellis, T.H.N. Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis. Plant J. 16, 643–650 (1998).

    Article  CAS  Google Scholar 

  24. Flavell, A.J. et al. A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach. Nucleic Acids Res. 31, e115 (2003).

    Article  Google Scholar 

  25. Shedlock, A.M. & Okada, N. SINE insertions: powerful tools for molecular systematics. Bioassays 22, 148–160 (2000).

    Article  CAS  Google Scholar 

  26. Pearce, S.R. et al. Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies. Plant J. 19, 711–717 (1999).

    Article  CAS  Google Scholar 

  27. Kalendar, R., Grob, T., Regina, M., Suoniemi, A. & Schulman, A.H. IRAP and REMAP: two new retrotransposon-based DNA fingerprinting techniques. Theor. Appl. Genet. 98, 704–711 (1999).

    Article  CAS  Google Scholar 

  28. Boyko, E., Kalendar, R., Korzun, V., Gill, B. & Schulman, A.H. Combined mapping of Aegilops tauschii by retrotransposon, microsatellite, and gene markers. Plant Mol. Biol. 48, 767–790 (2002).

    Article  CAS  Google Scholar 

  29. Manninen, O., Kalendar, R., Robinson, J. & Schulman, A.H. Application of BARE-1 retrotransposon markers to map a major resistance gene for net blotch in barley. Mol. Gen. Genet. 264, 325–334 (2000).

    Article  CAS  Google Scholar 

  30. Tanhuanpää, P. et al. Generation of SNP markers for short straw in oat (Avena sativa L.). Genome 49, 282–287 (2006).

    Article  Google Scholar 

  31. Antonius-Klemola, K., Kalendar, R. & Schulman, A.H. TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports. Theor. Appl. Genet. 112, 999–1008 (2006).

    Article  CAS  Google Scholar 

  32. Teo, C.H. et al. Genome constitution and classification using retrotransposon-based markers in the orphan crop banana. J. Plant Biol. 48, 96–105 (2005).

    Article  CAS  Google Scholar 

  33. Breto, M.P., Ruiz, C., Pina, J.A. & Asins, M.J. The diversification of Citrus clementina Hort. ex Tan., a vegetatively propagated crop species. Mol. Phylogenet. Evol. 21, 285–293 (2001).

    Article  CAS  Google Scholar 

  34. Pereira, H.S., Barao, A., Delgado, M., Morais-Cecilio, L. & Viegas, W. Genomic analysis of Grapevine Retrotransposon 1 (Gret 1) in Vitis vinifera. Theor. Appl. Genet. 111, 871–878 (2005).

    Article  CAS  Google Scholar 

  35. Smykal, P. Development of an efficient retrotransposon-based fingerprinting method for rapid pea variety identification. J. Appl. Genet. 47, 221–230 (2006).

    Article  Google Scholar 

  36. Baumel, A., Ainouche, M., Kalendar, R. & Schulman, A.H. Inter-retrotransposon amplified polymorphism (IRAP), and retotransposon-microsatellite amplified polymorphism (REMAP) in populations of the young allopolyploid species Spartina angelica Hubbard (Poaceae). Mol. Biol. Evol. 19, 1218–1227 (2002).

    Article  CAS  Google Scholar 

  37. Chadha, S. & Gopalakrishna, T. Retrotransposon-microsatellite amplified polymorphism (REMAP) markers for genetic diversity assessment of the rice blast pathogen (Magnaporthe grisea). Genome 48, 943–945 (2005).

    Article  CAS  Google Scholar 

  38. Díez, J., Béguiristain, T., Le Tacon, F., Casacuberta, J.M. & Tagu, D. Identification of Ty1-copia retrotransposons in three ectomycorrhizal basidiomycetes: evolutionary relationships and use as molecular markers. Curr. Genet. 43, 34–44 (2003).

    PubMed  Google Scholar 

  39. Schulman, A.H., Gupta, P.K. & Varshney, R.K. Organisation of retrotransposons and microsatellites in cereal genomes. in Cereal Genomics (eds. P.K. Gupta & R.K. Varshney) 83–118 (Kluwer, Dordrecht, The Netherlands, 2004).

    Google Scholar 

  40. Schulman, A.H., Flavell, A.J. & Ellis, T.H.N. The application of LTR retrotransposons as molecular markers in plants. Methods Mol. Biol. 260, 145–173 (2004).

    CAS  PubMed  Google Scholar 

  41. Tang, J.Q. et al. Alu-PCR combined with non-Alu primers reveals multiple polymorphic loci. Mamm. Genome 6, 345–349 (1995).

    Article  CAS  Google Scholar 

  42. Kovarova, M. & Draber, P. New specificity and yield enhancer of polymerase chain reactions. Nucleic Acids Res. 28, E70 (2000).

    Article  CAS  Google Scholar 

  43. Kalendar, R., Tanskanen, J., Immonen, S., Nevo, E. & Schulman, A.H. Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proc. Natl. Acad. Sci. USA 97, 6603–6607 (2000).

    Article  CAS  Google Scholar 

  44. Asins, M.J. et al. QTL analysis of citrus tristeza virus-citradia interaction. Theor. Appl. Genet. 108, 603–611 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

A.-M. Narvanto and S.U. Lönnqvist are thanked for their excellent technical assistance. The methods described here have been developed under grants from the Academy of Finland (ESGEMO, 6302016) and from the EU (TEBIODIV, GEDIFLUX, MMEDV), together with grants from CIMO of Finland, and in projects sponsored by Boreal Plant Breeding Ltd.

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Correspondence to Alan H Schulman.

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Kalendar, R., Schulman, A. IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat Protoc 1, 2478–2484 (2006). https://doi.org/10.1038/nprot.2006.377

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