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Analysis of 5S rDNA Genomic Organization Through the RepeatExplorer2 Pipeline: A Simplified Protocol

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Plant Cytogenetics and Cytogenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2672))

Abstract

The ribosomal RNA genes (rDNA) are universal genome components with a housekeeping function, given the crucial role of ribosomal RNA in the synthesis of ribosomes and thus for life-on-Earth. Therefore, their genomic organization is of considerable interest for biologists, in general. Ribosomal RNA genes have also been largely used to establish phylogenetic relationships, and to identify allopolyploid or homoploid hybridization.

Here, we demonstrate how high-throughput sequencing data, through graph clustering implemented in RepeatExplorer2 pipeline (https://repeatexplorer-elixir.cerit-sc.cz/galaxy/), can be helpful to decipher the genomic organization of 5S rRNA genes. We show that the linear shapes of cluster graphs are reminiscent to the linked organization of 5S and 35S rDNA (L-type arrangement) while the circular graphs correspond to their separate arrangement (S-type). We further present a simplified protocol based on the paper by (Garcia et al., Front Plant Sci 11:41, 2020) about the use of graph clustering of 5S rDNA homoeologs (S-type) to identify hybridization events in the species history. We found that the graph complexity (i.e., graph circularity in this case) is related to ploidy and genome complexity, with diploids typically showing circular-shaped graphs while allopolyploids and other interspecific hybrids display more complex graphs, with usually two or more interconnected loops representing intergenic spacers. When a three-genomic comparative clustering analysis from a given hybrid (homoploid/allopolyploid) and its putative progenitor species (diploids) is performed, it is possible to identify the corresponding homoeologous 5S rRNA gene families, and to elucidate the contribution of each putative parental genome to the 5S rDNA pool of the hybrid. Thus, the analysis of 5S rDNA cluster graphs by RepeatExplorer, together with information coming from other sources (e.g., morphology, cytogenetics) is a complementary approach for the determination of allopolyploid or homoploid hybridization and even ancient introgression events.

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References

  1. Van De Peer Y, Mizrachi E, Marchal K (2017) The evolutionary significance of polyploidy. Nat Rev Genet 18(7):411–424

    Article  PubMed  Google Scholar 

  2. Feliner GN, Casacuberta J, Wendel JF (2020) Genomics of evolutionary novelty in hybrids and polyploids. Front Genet 11:792

    Article  CAS  Google Scholar 

  3. Pedrosa-Harand A, de Almeida CCS, Mosiolek M, Blair M, Schweizer D, Guerra M (2006) Extensive ribosomal DNA amplification during Andean common bean (Phaseolus vulgaris L.) evolution. Theor Appl Genet 112(5):924–933

    Article  CAS  PubMed  Google Scholar 

  4. Mason AS, Wendel JF (2020) Homoelogous exchanges, segmental allopolyploidy, and polyploid genome evolution. Front Genet 11:1014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fehrer J, Slavikova R, Pastova L, Josefiova J, Mraz P, Chrtek J et al (2021) Molecular evolution and organization of ribosomal DNA in the Hawkweed Tribe (Cichorieae, Asteraceae). Front Plant Sci 12:647375

    Article  PubMed  PubMed Central  Google Scholar 

  6. Eilam T, Anikster Y, Millet E, Manisterski J, Feldman M (2008) Nuclear DNA amount and genome downsizing in natural and synthetic allopolyploids of the genera Aegilops and Triticum. Genome 51(8):616–627

    Article  CAS  PubMed  Google Scholar 

  7. Leitch IJ, Hanson L, Lim KY, Kovarik A, Chase MW, Clarkson JJ et al (2008) The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Ann Bot 101(6):805–814

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rieseberg LH, Ellstrand NC, Arnold M (1993) What can molecular and morphological markers tell us about plant hybridization? Crc Cr Rev Plant Sci 12(3):213–241

    CAS  Google Scholar 

  9. Twyford AD, Ennos RA (2012) Next-generation hybridization and introgression. Heredity 108(3):179–189

    Article  CAS  PubMed  Google Scholar 

  10. Alvarez I, Wendel JW (2003) Ribosomal ITS sequences and plant phylogenetic inference. Mol Phylogenet Evol 29:417–434

    Article  CAS  PubMed  Google Scholar 

  11. Feliner GN, Rosello JA (2007) Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Mol Phylogenet Evol 44(2):911–919

    Article  Google Scholar 

  12. Poczai P, Hyvonen J (2010) Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects. Mol Biol Rep 37(4):1897–1912

    Article  CAS  PubMed  Google Scholar 

  13. Cronn RC, Zhao X, Paterson AH, Wendel JF (1996) Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons. J Mol Evol 42(6):685–705

    Article  CAS  PubMed  Google Scholar 

  14. Volkov RA, Zanke C, Panchuk II, Hemleben V (2001) Molecular evolution of 5S rDNA of Solanum species (sect. Petota) application for molecular phylogeny and breeding. Theor Appl Genet 103(8):1273–1282

    Article  CAS  Google Scholar 

  15. Jang TS, McCann J, Parker JS, Takayama K, Hong SP, Schneeweiss GM et al (2016) rDNA loci evolution in the genus Glechoma (Lamiaceae). PLoS One 11(11):e0167177

    Article  PubMed  PubMed Central  Google Scholar 

  16. Volkov RA, Panchuk II, Borisjuk NV, Hosiawa-Baranska M, Maluszynska J, Hemleben V (2017) Evolution dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna. BMC Plant Biol 17(1):21

    Article  PubMed  PubMed Central  Google Scholar 

  17. Alexandrov OS, Razumova OV, Karlov GI (2021) A comparative study of 5S rDNA non-transcribed spacers in Elaeagnaceae species. Plants (Basel) 10(1):11

    Google Scholar 

  18. Cardoni S, Piredda R, Denk T, Grimm GW, Papageorgiou AC, Schulze ED et al (2022) 5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species. Plant J 109(4):909–926

    Article  CAS  PubMed  Google Scholar 

  19. Piredda R, Grimm GW, Schulze ED, Denk T, Simeone MC (2021) High-throughput sequencing of 5S-IGS in oaks: exploring intragenomic variation and algorithms to recognize target species in pure and mixed samples. Mol Ecol Resour 21(2):495–510

    Article  CAS  PubMed  Google Scholar 

  20. Volkov RA, Komarova NY, Hemleben V (2007) Ribosomal DNA in plant hybrids: inheritance, rearrangement, expression. Syst Biodivers 5(3):261–276

    Article  Google Scholar 

  21. Wendel JF, Schnabel A, Seelanan T (1995) Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proc Natl Acad Sci U S A 92(1):280–284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fulnecek J, Lim KY, Leitch AR, Kovarik A, Matyasek R (2002) Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species. Heredity 88(1):19–25

    Article  CAS  PubMed  Google Scholar 

  23. Weiss-Schneeweiss H, Tremetsberg K, Schneeweis GM, Parker JS, Stuessy TF (2008) Karyoptype diversification and evolution in diploid and polyploid South American Hypochaeris (Asteraceae) inferred from rDNA localization and genetic fingerprint data. Ann Bot 101(7):909–918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vozarova R, Herklotz V, Kovarik A, Tynkevich YO, Volkov RA, Ritz CM et al (2021) Ancient origin of two 5S rDNA families dominating in the genus Rosa and their behavior in the Canina-type meiosis. Front Plant Sci 12:643548

    Article  PubMed  PubMed Central  Google Scholar 

  25. Novak P, Neumann P, Pech J, Steinhaisl J, Macas J (2013) RepeatExplorer: a galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads. Bioinformatics 29(6):792–793

    Article  CAS  PubMed  Google Scholar 

  26. Novak P, Robledillo LA, Koblizkova A, Vrbobova I, Neumann P, Macas J (2017) TAREAN: a computational tool for identification and characterization of satellite DNA from unassembled short reads. Nucleic Acids Res 45(12):e111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Garcia S, Wendel JF, Borowska-Zuchowska N, Ainouche M, Kuderova A, Kovarik A (2020) The utility of graph clustering of 5S ribosomal DNA homoeologs in plant allopolyploids, homoploid hybrids and cryptic introgressants. Front Plant Sci 11:41

    Article  PubMed  PubMed Central  Google Scholar 

  28. Ellis TH, Lee D, Thomas CM, Simpson PR, Cleary WG, Newman MA et al (1988) 5S rRNA genes in Pisum: sequence, long range and chromosomal organization. Mol Gen Genet 214(2):333–342

    Article  CAS  PubMed  Google Scholar 

  29. Scoles CJ, Gill BSZ, Xin Y, Clarke BC, McIntyre CL, Chapman C et al (1988) Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of the Triticeae. Plant Syst Evol 160:105–122

    Article  CAS  Google Scholar 

  30. Gottlobmchugh SG, Levesque M, Mackenzie K, Olson M, Yarosh O, Johnson DA (1990) Organization of the 5S ribosomal-RNA genes in the soybean Glycine max (L.) merill and conservation of the 5S rDNA repeat structure in higher-plants. Genome 33(4):486–494

    Article  CAS  PubMed  Google Scholar 

  31. Schmidt T, Schwarzacher T, Heslopharrison JS (1994) Physical mapping of ribosomal-RNA genes by fluorescent in-situ hybridization and structural-analysis of 5S ribosomal-RNA genes and interfenic spacer sequences in sugar beet (Beta vulgaris). Theor Appl Genet 88(6–7):629–636

    Article  CAS  PubMed  Google Scholar 

  32. Capesius I (1997) Analysis of the ribosomal RNA gene repeat from the moss Funaria hygrometrica. Plant Mol Biol 33(3):559–564

    Article  CAS  PubMed  Google Scholar 

  33. Kawai H, Nakayama T, Inouye I, Kato A (1997) Linkage of 5S ribosomal DNA to other rDNAs in the chromophytic algae and related taxa. J Phycol 33(3):505–511

    Article  CAS  Google Scholar 

  34. Sone T, Fujisawa M, Takenaka M, Nakagawa S, Yamaoka S, Sakaida M et al (1999) Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants. Plant Mol Biol 41(5):679–685

    Article  CAS  PubMed  Google Scholar 

  35. Wicke S, Costa A, Muñoz J, Quandt D (2011) Restless 5S: the rearrangement(s) and evolution of the nuclear ribosomal DNA in land plants. Mol Phylogenet Evol 61(2):321–332

    Article  CAS  PubMed  Google Scholar 

  36. Matyasek R, Krumpolcova A, Lunerova J, Mikulaskova E, Rosello JA, Kovarik A (2019) Unique epigenetic features of ribosomal RNA genes (rDNA) in early diverging plant (Bryophytes). Front Plant Sci 10:3389

    Article  Google Scholar 

  37. Sousa A, Bechteler J, Temsch EM, Renner SS (2020) Different from tracheophytes, liverworts commonly have mixed 35S and 5S arrays. Ann Bot 125(7):1057–1064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Garcia S, Lim KY, Chester M, Garnatje T, Pellicer J, Valles J et al (2009) Linkage of 35S and 5S rRNA genes in Artemisia (family Asteraceae): first evidence from angiosperms. Chromosoma 118(1):85–97

    Article  CAS  PubMed  Google Scholar 

  39. Garcia S, Panero JL, Siroky J, Kovarik A (2010) Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family. BMC Plant Biol 10:176

    Article  PubMed  PubMed Central  Google Scholar 

  40. Mazzella C, Rodriguez M, Vaio M, Gaiero P, López-Carro B, Santinaque FF et al (2010) Karyological features of Achyrocline (Asteraceae, Gnaphalieae): stable karyotypes, low DNA content variation and linkage of rRNA genes. Cytogenet Genome Res 128:169–176

    Article  CAS  PubMed  Google Scholar 

  41. Galian JA, Rosato M, Rosello JA (2012) Early evolutionary colonization of the nuclear ribosomal 5S and 45S gene families in seed plants: evidence from the living fossil gymnosperm Ginkgo biloba. Heredity 108(6):640–646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Garcia S, Kovarik A, Leitch AR, Garnatje T (2017) Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database. Plant J 89(5):1020–1030

    Article  CAS  PubMed  Google Scholar 

  43. Röser M, Winterfeld G, Grebenstein B, Hemleben V (2001) Molecular diversity and physical mapping of 5S rDNA in wild, and cultivated oat grasses (Poaceae: Aveneae). Mol Phylogenet Evol 21(2):198–217

    Article  PubMed  Google Scholar 

  44. Garcia S, Kovarik A (2013) Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation. Heredity 111(1):23–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang W, Wan T, Becher H, Kuderova A, Leitch IJ, Garcia S et al (2019) Remarkable variation of ribosomal DNA organization and copy number in gnetophytes: a distinct lineage of gymnosperms. Ann Bot 123(5):767–781

    Article  CAS  PubMed  Google Scholar 

  46. Heitkam T, Petrasch S, Zakrzewski F, Kogler A, Wenke T, Wanke S et al (2015) Next-generation sequencing reveals differentially amplified tandem repeats as a major genome component of Northern Europe’s oldest Camellia japonica. Chromosom Res 23(4):791–806

    Article  CAS  Google Scholar 

  47. Novak P, Neumann P, Macas J (2020) Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2. Nat Protoc 15:3745–3776

    Article  CAS  PubMed  Google Scholar 

  48. Vitales D, Garcia S, Dodsworth S (2020) Reconstructing phylogenetic relationships based on repeat sequences similarities. Mol Phylogenet Evol 147:106766

    Article  PubMed  Google Scholar 

  49. Herklotz V, Kovarik A, Wissemann V, Lunerova J, Vozarova R, Buschmann S et al (2021) Power and weakness of repetition – evaluating the phylogenetic signal from repeatomes in the family Rosaceae with two case studies from genera Prone to polyploidy and hybridization (Rosa and Fragaria). Front Plant Sci 12:738119

    Article  PubMed  PubMed Central  Google Scholar 

  50. Wang WC, Ma L, Becher H, Garcia S, Kovarikova A, Leitch IJ et al (2016) Astonishing 35S rDNA diversity in the gymnosperm species Cycas revolutiva Thunb. Chromosoma 125(4):683–699

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Institut Botànic de Barcelona (CSIC – Ajuntament de Barcelona), Barcelona, Spain

    Sònia Garcia & Joan Pere Pascual-Díaz

  2. Department of Molecular Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic

    Alice Krumpolcová & Ales Kovarík

  3. Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

    Alice Krumpolcová

Authors
  1. Sònia Garcia
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  2. Joan Pere Pascual-Díaz
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  3. Alice Krumpolcová
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  4. Ales Kovarík
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Correspondence to Ales Kovarík .

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Editors and Affiliations

  1. Institute of Botany, TU Dresden, Dresden, Germany

    Tony Heitkam

  2. Botanical Institute of Barcelona, Barcelona, Spain

    Sònia Garcia

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Garcia, S., Pascual-Díaz, J.P., Krumpolcová, A., Kovarík, A. (2023). Analysis of 5S rDNA Genomic Organization Through the RepeatExplorer2 Pipeline: A Simplified Protocol. In: Heitkam, T., Garcia, S. (eds) Plant Cytogenetics and Cytogenomics. Methods in Molecular Biology, vol 2672. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3226-0_30

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  • DOI: https://doi.org/10.1007/978-1-0716-3226-0_30

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  • Publisher Name: Humana, New York, NY

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