title: Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae).
abstract: OBJECTIVE
  Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids. Medicago arborea is a tetraploid species that has a single 45S rDNA locus. This feature makes this species a suitable case study to assess the fate of ribosomal IGS homogenization in polyploid species showing nucleolus organizer region (NOR) reduction.
  
  
  METHODS
  The intergenic spacer (IGS) region was amplified by long PCR and the fragments were cloned and sequenced by a primer-walking strategy. The physical mapping of the whole and partial IGS variants was assessed by fluorescent in situ hybridization (FISH) and fibre-FISH methods on mitotic chromosomes and extended DNA fibres, respectively.
  
  
  RESULTS
  Two IGS fragments of 4·8 and 3·5 kb were obtained showing structural features of functional sequences. The shorter variant appears to be a truncated copy of the 4·8 kb fragment that lacks the duplication of the transcription initiation site region and the entire D region. The physical localization of the two IGS variants on metaphase chromosomes and extended DNA fibres using FISH corroborated their joint presence within the same locus. In addition, no spatial structure of the two variants was detected within the NOR.
  
  
  CONCLUSIONS
  The results suggest that full sequence homogenization is not operating within the NOR locus of M. arborea. The structure of the NOR locus reported here departs from the models of IGS heterogeneity present in plants and caution against assuming the widespread belief that intragenomic ribosomal heterogeneity is mainly due to sequence variation between paralogous loci.
authors.affiliation: Jardín Botánico, ICBiBE, Universidad de Valencia, c/Quart 80, E-46008, Valencia, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Galián
authors.affiliation: Jardín Botánico, ICBiBE, Universidad de Valencia, c/Quart 80, E-46008, Valencia, Spain.
authors.forename: Marcela
authors.initials: M
authors.lastname: Rosato
authors.affiliation: Jardín Botánico, ICBiBE, Universidad de Valencia, c/Quart 80, E-46008, Valencia, Spain Marimurtra Bot. Garden, Carl Faust Fdn, PO Box 112, E-17300 Blanes, Catalonia, Spain rossello@uv.es.
authors.forename: Josep A
authors.initials: JA
authors.lastname: Rosselló
mesh_terms: D032461:Chromosomes, Plant / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D014644:Genetic Variation; D018745:Genome, Plant / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D029902:Medicago / Q000235:genetics*; D005810:Multigene Family; D009697:Nucleolus Organizer Region / Q000235:genetics*; D011123:Polyploidy; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer
keywords: 45S rDNA; Fabaceae; IGS; Medicago arborea; fibre-FISH; intergenic spacer; intragenomic variation; nuclear ribosomal cistron; polyploidy; transcription initiation site