Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

Abstract

Background: In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in approximately 200 species representing the family diversity and other closely related groups.

Results: Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes.

Conclusions: Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units, their copy number and chromosomal organisation may occur within relatively short evolutionary time. We hypothesize that the 5S gene integration within the 35S unit might have repeatedly occurred during plant evolution, and probably once in Asteraceae.

Figure 1

PCR analysis of rDNA arrangements. The upper panel (A) illustrates the strategy used for detection of mutual 26S and 5S positions. The position and orientation of primers are indicated by arrows. Grey boxes indicate coding sequences. "B", cutting sites for BamHI. The bottom panel (B) shows four profiles of PCR products obtained. "s", electrophoresis start.

Figure 2

Southern blot hybridisation analysis of rDNA arrangements. The genomic DNAs representative species from each Group (I-IV) were digested with BamHI and hybridised on blots with the respective DNA probes. The images were cut and arranged into panels, each representing individual species hybridised with the 5S and 26S probes. The sizes of monomeric units of tandemly arranged 5S repeats (Groups I, III and IV) were ~0.5 kb; the size of a major 26S-5S band in Group II species was 2-3 kb.

Figure 3

Quantification of the linked rRNA genes copy number by real time PCR. The Pr1-5SLf primer set (Figure 1A) was used to amplify inversely positioned 26S-5S genes. The graph shows mean values obtained from three independent experiments. Example of the amplification plot for Artemisia and Elachanthemum is shown in Additional file 2.

Figure 4

The position and organisation of 5S insertions within the 26S-18S intergenic spacer. (A). The 26S, 18S and 5S genic regions are in green and red boxes, respectively. Mutated or incomplete 5S copies are indicated by vertical strips. The sizes of IGS1 are depicted with thin lines above the units. Direction of transcription is illustrated by thick arrows below the genes. Arrowheads indicate sequences homologous to terminal inverted repeats (TIR) of Cassandra elements, aligned in (B). (*) the IGS type with a single 5S gene is the most abundant in A. absinthium; (**) species with typical unlinked arrangement of units.

Figure 5

Alignment of 5S genic regions and flanking sequences. Conserved regulatory motifs essential for the polymerase III transcription [51] are highlighted. The insertions with complete and incomplete 5S copies are separated by a horizontal line. Note the highly conserved genic regions are well separated from the highly diverged 5' and 3' sequence ends. The 3' flanking sequences are only partially aligned because of the divergence. The numbers after the species name in titles indicate clones. TATA - TATA box; TSS - transcription start site; IE - internal regulatory element; Termin. - transcription terminator. The bottom strands (with respect to the 26S gene transcription) are shown except for Madia sativa. Underlined solid line - a motif resembling reverse transcriptase primer binding site (methionine tRNA). Underlined dashed line - a motif resembling a polyadenylation site.

Figure 6

Phylogenetic analysis of the 5S genic (A) and intergenic spacer 1 (IGS1) sequences (B). The arrangement of rRNA genes is depicted by U (unlinked) and L (linked). The phylogenetic positions are based on the nearest neighbor analysis of multiple alignments. Accession numbers of taxa are given in Additional file 4. The "5S" and "Cass." symbols before the taxon name indicate either 5S genic or Cassandra sequences. Numbers above branches indicate bootstrap percentages >70. The Saccharomyces cereviseae 5S gene was used as outgroup in (A). The IGS1 cladogram is an unrooted tree. Scale bars indicate the number of base substitutions per site.

Figure 7

Fluorescent in situ hybridisation of metaphase chromosomes and interphase nuclei of species that evolved linked arrangement of rRNA genes: (A-C) Tagetes patula (2n = 48); (D-F) Helichrysum bracteatum (2n = 22); (G-I) Tripleurospermum maritimum (2n = 36), (J-L) Coreopsis major (2n = 24). The 35S and 5S loci are labelled in green and red, respectively. Arrows indicate a possible solo 5S locus in Coreopsis major.

Figure 8

Fluorescent in situ hybridisation of metaphase chromosomes and interphase nuclei of species that evolved unlinked arrangement of rRNA genes. The individual slides show merged 26S and 5S signals: (A) Dahlia pinnatta (2n = 64), (B) Helianthus annuus (2n = 34); (C) Chrysanthemum zawadskii (2n = 54), (D) Aster alpinus (2n = 18); (E) Calendula officinalis (2n = 28); (F) Tragopogon mirus (2n = 24). Note that the 26S loci are mostly terminal whereas the 5S sites are mostly interstitial. Arrowheads in (A) indicate juxtaposition of 35S and 5S arrays without physical linkage of units.

Figure 9

Phylogenetic distribution of rDNA arrangement in the subfamily Asteroideae. All groups showing homogenised linked arrangement are present. Tree topology is according to the published phylogeny [21]. Red and black lines indicate evolutionary trajectories of linked and unlinked units, respectively.

Figure 10

Hypothetical model illustrating a cascade of events accompanying 5S rDNA evolution in Asteraceae. (A) Mobilisation of 5S genes by a retrotransposition event and their integration within the 35S unit; (B) short (< 100 units) tandems of 35S-5S repeats generated by intralocus non-homologous recombination; inactivation of retroelement activity by mutation and/or epigenetic silencing. (C) amplification and spreading of 35S-5S arrays across the chromosomes mediated by mechanisms such rolling circle replication and reintegration of covalently linked circles. Loss of arrays of unlinked units. Green rectangles - units of 35S genes, red rectangles - units of 5S genes. Dotted line depicts the reversion process from linked to unlinked genotype. For the simplicity, proportions of gene sizes are not in scale.