Skip to main content
Log in

Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.

  • Original Article
  • Published:
Protoplasma Aims and scope Submit manuscript

Abstract

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA+ bands were colocalized with the 45S sites.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Barker RE, Kilgore JA, Cook RL, Garay AE, Warnke SE (2001) Use of flow cytometry to determine ploidy level of ryegrass. Seed Sci Tech 29:493–502

    Google Scholar 

  • Barros E, Silva AE, Guerra M (2009) The meaning of DAPI bands observed after C-banding and FISH procedures. Biotech Histochem 85:115–125

    Article  Google Scholar 

  • Biessmann H, Mason JM (1992) Functional reconstitution of wild-type and mutant Tetrahymena telomerase. Adv Genet 30:185–249

    Article  CAS  PubMed  Google Scholar 

  • Blackburn EH (1991) Structure and function of telomeres. Nature 350:569–573

    Article  CAS  PubMed  Google Scholar 

  • Boulon S, Westman BJ, Hutten S, Boisvert F, Al L (2010) The nucleolus under stress. Mol Cell 40:216–227

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bustamante FO, Techio VH, Rocha LC, Torres GA, Davide LC, Mittelmann A (2014) Distribution of rDNA in diploid and poliploid Lolium multiflorum Lam. reveals fragile sites in 45S rDNA region. Crop Sci 54:1–9

    Article  Google Scholar 

  • Brown JD, O’Neill RJ (2010) Chromosomes, conflict, and epigenetics: chromosomal speciation revisited. Annu Rev Genom Hum G 11:291–316

  • Buttel I, Fechter A, Schwab M (2004) Common fragile sites and cancer: targeted cloning by insertional mutagenesis. Ann NY Acad Sci 1028:14–27

    PubMed  Google Scholar 

  • Caperta AD, Neves N, Morais-Cecílio L, Malhó R, Viegas W (2002) Genome restructuring in rye affects the expression, organization and disposition of homologous rDNA loci. J Cell Sci 115:2839–2846

    CAS  PubMed  Google Scholar 

  • Carnide V, Orellana J (1986) Analysis of nucleolar organizer regions by silver staining in two Lolium species. Euphytica 35:503–507

    Article  Google Scholar 

  • Datson PM, Murray BG (2006) Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism? Chromosom Res 14:845–857

    Article  CAS  Google Scholar 

  • Dong F, Song J, Naess SK, Helgeson JP, Gebhardt C, Jiang J (2000) Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato. Theor Appl Genet 101:1001–1007

    Article  CAS  Google Scholar 

  • Dubcovsky J, Dvorak J (1995) Ribosomal-RNA multigene loci-nomads of the Triticeae genomes. Genetics 140:1367–1377

    PubMed Central  CAS  PubMed  Google Scholar 

  • Glover TW (2006) Common fragile sites. Cancer Lett 232:4–12

    Article  CAS  PubMed  Google Scholar 

  • Guerra M (1986) Reviewing the chromosome nomenclature of Levan et al. Braz J Genet 9:741–743

    Google Scholar 

  • Guerra M, Souza MJ (2002) Como observar cromossomos: um guia de técnicas em citogenética vegetal, animal e humana. FUNPEC, Ribeirão Preto

    Google Scholar 

  • Hoshi Y, Plader W, Malepszy S (1998) Physical mapping of 45S rRNA gene loci in the cucumber (Cucumis sativus L.) using fluorescence in situ hybridization. Caryologia 52:49–57

    Article  Google Scholar 

  • Huang J, Ma L, Yang F, Fei Y, Fei S, Li L (2008) 45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp. PLoS One 3:1–7

    CAS  Google Scholar 

  • Huang J, Ma L, Sundararajan S, Fei S, Li L (2009) Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne. Protoplasma 236:59–65

    Article  CAS  PubMed  Google Scholar 

  • Huang M, Li H, Zhang L, Gao F, Wang P, Hu Y, Yan S, Zhao L, Zhang Q, Tan J, Liu X, He S, Li L (2012) Plant 45S rDNA clusters are fragile sites and their instability is associated with epigenetic alterations. PLoS One:7 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035139. Accessed 14 August 2013

  • Huang S, Rothblum L, Chen D (2010) Ribosomal chromatin: the meaning of DAPI bands observed after C-banding and FISH procedures. Biotech Histochem 85:115–125

    Article  Google Scholar 

  • Jauhar PP (1993) Alien gene transfer and genetic enrichment of bread wheat. In: Damania AB (ed) Biodiversity and wheat improvement, A. Wiley-Sayce, Ciudad del Mexico, pp 103–104

    Google Scholar 

  • Jiang J, Gill BS (1994) New 18S-26S ribosomal RNA gene loci: chromosomal and marks for the evolution of polyploidy wheats. Chromosoma 103:179–185

    Article  CAS  PubMed  Google Scholar 

  • Jiang JM, Gill BS, Wang GL, Ronald PC, Ward DC (1995) Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc Natl Acad Sci U S A 92:4487–4491

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jones C, Penny L, Mattina T, Yu S, Baker E, Voullaire L, Langdon WY, Sutherland GR, Richards RI, Tunnacliffe A (1995) Association of a chromosome deletion syndrome with a fragile site within the proto-oncogene CBL2. Nature 376:145–149

    Article  CAS  PubMed  Google Scholar 

  • Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27:247–254

    Article  CAS  PubMed  Google Scholar 

  • King K, Torres RA, Zentgraf U, Hemleben V (1993) Molecular evolution of the intergenic spacer in the nuclear ribosomal RNA genes of Cucurbitaceae. J Mol Evol 36:144–152

    Article  CAS  PubMed  Google Scholar 

  • Ksiazczyk T, Taciak M, Zwierzykowski Z (2010) Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH. J Appl Genet 51:449–460

    Article  CAS  PubMed  Google Scholar 

  • Lideikytë L, Pašakinskienė I, Lemežienė N, Nekrošas S, Kanapeckas J (2008) FISH assessment of ribosomal DNA sites in the chromosome sets of Lolium, Festuca and Festulolium. Agriculture 265:116–124

    Google Scholar 

  • Malik CP, Thomas PT (1966) Karyotipic studies in some Lolium and Festuca species. Caryologia 19:167–196

    Article  Google Scholar 

  • Mcstay B (2006) Nucleolar dominance: a model for rRNA gene silencing. Gene Dev 20:1207–1214

    Article  CAS  PubMed  Google Scholar 

  • Montijn MB, Houtsmuller AB, Hoopen RT, Oud JL, Nanninga N (1999) The 5S rRNA gene clusters have a defined orientation toward the nucleolus in Petunia hybrid and Crepis capillaris. Chromosom Res 7:387–399

    Article  CAS  Google Scholar 

  • Ozeri-Galai E, Schwartz M, Rahat A, Kerem B (2008) Interplay between ATM and ATR in the regulation of common fragile site stability. Oncogene 27:2109–2117

    Article  CAS  PubMed  Google Scholar 

  • Pedrosa A, Sandal N, Stougaard J, Schweizer D, Bachmair A (2002) Chromosomal map of the model legume Lotus japonicas. Genetics 161:1661–1672

    PubMed Central  CAS  PubMed  Google Scholar 

  • Pedrosa A, Schweizer D, Guerra M (2000) Cytological heterozygosity and the hybrid origin of sweet orange [Citrus sinensis (L.) Osbeck]. Theor Appl Genet 100:361–367

    Article  Google Scholar 

  • Pérez R, De Bustos A, Jouve N, Cuadrado A (2009) Localization of Rad50, a single-copy gene, on group 5 chromosomes of wheat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH). Cytogenet Genome Res 125:321–328

    Article  PubMed  Google Scholar 

  • Phillips RL (1978) Molecular cytogenetics of the nucleolus organizer region. In: WALDEN DB (ed) Maize breeding and genetics. Wiley, New York, pp 711–742

    Google Scholar 

  • Polok K (2007) Molecular evolution of the genus Lolium sp. Studio Poligrafii Komputerowej, Olsztyn

    Google Scholar 

  • Raskina O, Belyayev A, Nevo E (2004a) Activity of the En/Spm-like transposons in meiosis as a base for chromosome repatterning in a small, isolated, peripheral population of Aegilops speltoides Tausch. Chromosom Res 12:153–161

    Article  CAS  Google Scholar 

  • Raskina O, Belyayev A, Nevo E (2004b) Quantum speciation in Aegilops: molecular cytogenetic evidence from rDNA cluster variability in natural populations. Proc Natl Acad Sci U S A 101:14818–14823

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Richards EJ, Ausubel FM (1988) Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53:127–136

    Article  CAS  PubMed  Google Scholar 

  • Richards RI (2001) Fragile and unstable chromosomes in cancer: causes and consequences. Trends Genet 17:339–345

    Article  CAS  PubMed  Google Scholar 

  • Roa F, Guerra M (2012) Distribution of 45S rDNA sites in chromosomes of plants: structural and evolutionary implications. BMC Evol Biol 12:255

    Article  Google Scholar 

  • Rogers SO, Bendich AJ (1987) Ribosomal RNA genes in plants: variability in copy number in an intergenic spacer. Plant Mol Biol 9:509–520

    Article  CAS  PubMed  Google Scholar 

  • Ruiz-Herrera A, Robinson TJ (2007) Afrotherian fragile sites, evolutionary breakpoints and phylogenetic inference from genomic assemblies. BMC Evol Biol 7:1–15

    Article  Google Scholar 

  • Schubert I, Kunzel G (1990) Position-dependent NOR activity in barley. Chromosoma 99:352–359

    Article  Google Scholar 

  • Schubert I, Wobus U (1985) In situ hybridization confirms jumping nucleolus organizing regions in Allium. Chromosoma 92:143–148

    Article  Google Scholar 

  • Schwartz G, Zlotorynski E, Goldberg M, Ozeri E, Rahat A, Sage C, Chen BPC, Chen DJ, Agami R, Kerem B (2005) Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability. Gene Dev 19:2715–2726

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Taketa S, Ando H, Takeda K, Ichii M, Bothmer RV (2001) Physical locations of 5S and 18S-25S rDNA in Asian and American diploid Hordeum species with the I genome. Heredity 86:522–530

    Article  CAS  PubMed  Google Scholar 

  • Taketa S, Ando H, Takeda K, Ichii M, Bothmer RV (2005) Ancestry of American polyploidy Hordeum species with the I genome inferred from 5S and 18S-25S rDNA. Ann Bot 96:23–33

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Terrel EE (1968) A taxonomic revision of the genus Lolium. Technical Bulletins, USDA

    Google Scholar 

  • Thomas HM (1981) The Giemsa C-band karyotypes of six Lolium species. Heredity 46:263–267

    Article  CAS  Google Scholar 

  • Thomas HM, Harper JA, Meredith MR, Morgan WG, Thomas ID, Timms E, King IP (1996) Comparasion of ribosomal DNA sites in Lolium species by fluorescence in situ hybridization. Chromosom Res 4:486–490

    Article  CAS  Google Scholar 

  • Thomas HM, Harper JA, Morgan WG (2001) Gross chromosome rearrangements are occurring in an accession of the grass Lolium rigidum. Chromosom Res 9:585–590

    Article  CAS  Google Scholar 

  • Vaio M, Speranza P, Valls JF, Guerra M, Mazzella C (2005) Localization of the 5S and 45S rDNA sites and cpDNA sequence analysis in species of the Quadrifaria Group of Paspalum (Poaceae, Paniceae). Ann Bot 96:191–200

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang Y, Griffith J (1996) Methylation of expanded CCG triplet repeat DNA from fragile X syndrome patients enhances nucleosome exclusion. J Biol Chem 271:22937–22940

    Article  CAS  PubMed  Google Scholar 

  • Winterfeld G, Röser M (2007a) Chromosomal localization and evolution of satellite DNAs and heterochromatin in grasses (Poaceae), especially tribe Aveneae. Pl Syst Evol 264:75–100

    Article  CAS  Google Scholar 

  • Winterfeld G, Röser M (2007b) Disposition of ribosomal DNAs in the chromosomes of perennial oats (Poaceae: Aveneae). Bot J Linn Soc 155:193–210

    Article  Google Scholar 

  • Yamada T, Forster JW, Humphreys MW, Takamizo T (2005) Genetics and molecular breeding in Lolium/Festuca. Grassl Sci 51:89–106

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank the Brazilian National Council for Scientific and Technological Development—CNPq, Foundation for Research Support of the State of Minas Gerais—FAPEMIG, Coordination for the Improvement of Higher Education Personnel—CAPES for the financial support to research and scholarship and M.S. Luiz Carlos de Almeida Rodrigues for contribution in preparing the figures.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Vânia Helena Techio.

Additional information

Handling Editor: Peter Nick

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rocha, L.C., de Oliveira Bustamante, F., Silveira, R.A.D. et al. Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.. Protoplasma 252, 451–460 (2015). https://doi.org/10.1007/s00709-014-0690-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00709-014-0690-4

Keywords

Navigation