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Transposon-Based Tagging: IRAP, REMAP, and iPBS

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Molecular Plant Taxonomy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1115))

Abstract

Retrotransposons are a major component of virtually all eukaryotic genomes, which makes them useful as molecular markers. Various molecular marker systems have been developed that exploit the ubiquitous nature of these genetic elements and their property of stable integration into dispersed chromosomal loci that are polymorphic within species. To detect polymorphisms for retrotransposon insertions, marker systems generally rely on PCR amplification between the retrotransposon termini and some component of flanking genomic DNA. The main methods of IRAP, REMAP, RBIP, and SSAP all detect the polymorphic sites at which the retrotransposon DNA is integrated into the genome. Marker systems exploiting these methods can be easily developed and are inexpensively deployed in the absence of extensive genome sequence data. Here, we describe protocols for the IRAP, REMAP, and iPBS techniques, including methods for PCR amplification with a single primer or with two primers, and agarose gel electrophoresis of the product using optimal electrophoresis buffers; we also describe iPBS techniques for the rapid isolation of retrotransposon termini and full-length elements.

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References

  1. Wicker T, Keller B (2007) Genome-wide comparative analysis of copia retrotransposons in Triticeae, rice, and Arabidopsis reveals conserved ancient evolutionary lineages and distinct dynamics of individual copia families. Genome Res 17:1072–1081

    Article  CAS  PubMed  Google Scholar 

  2. Cheng ZJ, Murata M (2003) A centromeric tandem repeat family originating from a part of Ty3/gypsy-retroelement in wheat and its relatives. Genetics 164:665–672

    CAS  PubMed  Google Scholar 

  3. Boyko E et al (2002) Combined mapping of Aegilops tauschii by retrotransposon, microsatellite, and gene markers. Plant Mol Biol 48:767–790

    Article  CAS  PubMed  Google Scholar 

  4. Lamb JC et al (2007) Plant chromosomes from end to end: telomeres, heterochromatin and centromeres. Curr Opin Plant Biol 10:116–122

    Article  CAS  PubMed  Google Scholar 

  5. Meyer W et al (1993) Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans. J Clin Microbiol 31:2274–2280

    CAS  PubMed Central  PubMed  Google Scholar 

  6. Sivolap IM, Kalendar RN, Chebotar SV (1994) The genetic polymorphism of cereals demonstrated by PCR with random primers. Tsitol Genet 28:54–61

    CAS  PubMed  Google Scholar 

  7. Charlieu JP et al (1992) 3′ Alu PCR: a simple and rapid method to isolate human polymorphic markers. Nucleic Acids Res 20: 1333–1337

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Sabot F, Schulman AH (2006) Parasitism and the retrotransposon life cycle in plants: a hitchhiker’s guide to the genome. Heredity 97: 381–388

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Hedges DJ, Batzer MA (2005) From the margins of the genome: mobile elements shape primate evolution. Bioessays 27:785–794

    Article  CAS  PubMed  Google Scholar 

  13. Ostertag EM, Kazazian HH (2005) Genetics: LINEs in mind. Nature 435:890–891

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. Bannert N, Kurth R (2004) Retroelements and the human genome: new perspectives on an old relation. Proc Natl Acad Sci U S A 101:14572–14579

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Kalendar R et al (2010) Analysis of plant diversity with retrotransposon-based molecular markers. Heredity 106:520–530

    Article  PubMed  Google Scholar 

  18. Waugh R et al (1997) 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

    Article  CAS  PubMed  Google Scholar 

  19. Vogel JM, Morgante M (1992) A microsatellite-based, multiplexed genome assay, In Plant Genome III Conference. San Diego, CA USA

    Google Scholar 

  20. Korswagen HC et al (1996) Transposon Tc1-derived, sequence-tagged sites in Caenorhabditis elegans as markers for gene mapping. Proc Natl Acad Sci U S A 93: 14680–14685

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Van den Broeck D et al (1998) Transposon Display identifies individual transposable elements in high copy number lines. Plant J 13:121–129

    PubMed  Google Scholar 

  22. Ellis THN 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 260:9–19

    CAS  PubMed  Google Scholar 

  23. Vos P et al (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 21:4407–4414

    Article  Google Scholar 

  24. Yu G-X, Wise RP (2000) An anchored AFLP- and retrotransposon-based map of diploid Avena. Genome 43:736–749

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  26. Lee D et al (1990) A copia-like element in Pisum demonstrates the uses of dispersed repeated sequences in genetic analysis. Plant Mol Biol 15:707–722

    Article  CAS  PubMed  Google Scholar 

  27. Syed NH, Flavell AJ (2006) Sequence-specific amplification polymorphisms (SSAPs): a multi-locus approach for analyzing transposon insertions. Nat Protoc 1:2746–2752

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Vershinin AV, Ellis TH (1999) Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea. Mol Gen Genet 262:703–713

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  31. Hirochika H, Hirochika R (1993) Ty1-copia group retrotransposons as ubiquitous components of plant genomes. Jpn J Genet 68:35–46

    Article  CAS  PubMed  Google Scholar 

  32. Flavell AJ et al (1992) Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acids Res 20:3639–3644

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Ellis THN et al (1998) Ty1-copia class retrotransposon insertion site polymorphism for linkage and diversity analysis in pea. Mol Gen Genet 260:9–19

    CAS  PubMed  Google Scholar 

  34. Kalendar R et al (2008) Cassandra retrotransposons carry independently transcribed 5S RNA. Proc Natl Acad Sci U S A 105:5833–5838

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Witte CP et al (2001) Terminal-repeat retrotransposons in miniature (TRIM) are involved in restructuring plant genomes. Proc Natl Acad Sci U S A 98:13778–13783

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Corpet F (1988) Multiple sequence alignment with hierarchical-clustering. Nucleic Acids Res 16:10881–10890

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes, Genomes and Genomics 3:1–14

    Google Scholar 

  38. Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Baumel A et al (2002) Inter-retrotransposon amplified polymorphism (IRAP), and retrotransposon-microsatellite amplified polymorphism (REMAP) in populations of the young allopolyploid species Spartina angelica Hubbard (Poaceae). Mol Biol Evol 19:1218–1227

    Article  CAS  PubMed  Google Scholar 

  41. Boronnikova SV, Kalendar RN (2010) Using IRAP markers for analysis of genetic variability in populations of resource and rare species of plants. Russ J Genet 46:36–42

    Article  CAS  Google Scholar 

  42. Belyayev A et al (2010) Transposable elements in a marginal plant population: temporal fluctuations provide new insights into genome evolution of wild diploid wheat. Mob DNA 1:6

    Article  PubMed Central  PubMed  Google Scholar 

  43. Smýkal P et al (2011) Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers. Theor Appl Genet 122:1385–1397

    Article  PubMed  Google Scholar 

  44. Manninen OM et al (2006) Mapping of major spot-type and net-type net-blotch resistance genes in the Ethiopian barley line CI 9819. Genome 49:1564–1571

    Article  CAS  PubMed  Google Scholar 

  45. Tang JQ et al (1995) Alu-PCR combined with non-Alu primers reveals multiple polymorphic loci. Mamm Genome 6:345–349

    Article  CAS  PubMed  Google Scholar 

  46. Shedlock AM, Okada N (2000) SINE insertions: powerful tools for molecular systematics. Bioessays 22:148–160

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Academy of Finland grant 134079. Anne-Mari Narvanto is thanked for excellent technical assistance.

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Kalendar, R., Schulman, A.H. (2014). Transposon-Based Tagging: IRAP, REMAP, and iPBS. In: Besse, P. (eds) Molecular Plant Taxonomy. Methods in Molecular Biology, vol 1115. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-767-9_12

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  • DOI: https://doi.org/10.1007/978-1-62703-767-9_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-766-2

  • Online ISBN: 978-1-62703-767-9

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