Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids
- Roman Matyášek†1,
- Simon Renny-Byfield†2, 3,
- Jaroslav Fulneček1,
- Jiří Macas4,
- Marie-Angele Grandbastien5,
- Richard Nichols2,
- Andrew Leitch2 and
- Aleš Kovařík1Email author
© Matyášek et al.; licensee BioMed Central Ltd. 2012
Received: 24 September 2012
Accepted: 13 December 2012
Published: 23 December 2012
Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units.
We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci.
Results and Conclusions
In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations.
In most eukaryotes, 5S and 18–5.8–26S nuclear ribosomal DNA (rDNA) units occur in tandem array at one or several loci. Each large rDNA unit (designated 45S and 35S for animals and plants, respectively) contains three different rRNA genes (18S, 5.8S and 26S), which are separated by two internal transcribed spacer sequences (ITS1 and ITS2). Each group is separated by the intergenic spacer (IGS, ). The genic regions are highly conserved, whereas ITS divergence is sufficient to resolve species relationships within most genera. The IGS, which contains the transcription-start site and genetic and epigenetic features that influence the regulation of downstream genes, diverges more rapidly than the ITS.
Of particular interest to evolutionary biologists is the pattern of divergence of the whole rDNA array known as concerted evolution, in which the units of the rDNA array are very similar within a species but diverge between species. This pattern suggests that the arrays are subject to homogenisation – so that novel variants arising by mutation spread relatively rapidly along the array within any one species [2–4]. Computer modelling has suggested homogenisation would act to reduce mutational load and could therefore be favoured by selection [5, 6]. An alternative model of multigene family evolution, perhaps most generally applicable to small multigene families, argues for birth and death of duplicate gene copies associated with selection, explaining relatively high diversity observed in gene families [7, 8]. It has been suggested that this model might explain the unusually high levels of ITS and IGS polymorphisms in some species (up to 40% in some cases) [9–11].
In angiosperms, rDNA evolution has been challenging to study, since there are many copies of the large 35S unit, amounting to thousands, or tens of thousands of units  distributed over one or several chromosomal loci . High copy-number and sequence conservation of tandemly-arranged units make the analysis of the structure and organisation of the entire array difficult by classical cloning and sequencing approaches. Consequently, rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been comprehensively studied in any organism carrying more than a few hundred copies of the 35S rDNA unit. The advent of next generation sequencing (NGS) allows such an analysis for the first time [14, 15]; in this study we therefore took advantage of NGS to explore rDNA homogeneity in four angiosperm species in the genus Nicotiana (Solanaceae).
Nicotiana is an ideal choice for the study of rDNA variation because we can take advantage of existing studies on the evolutionary processes operating across the genus. These studies at the cytogenetic [16–18], molecular [19, 20] and genomic [21–23] levels have given insights into species relationships and patterns of species divergence. There are several phylogenetic studies examining patterns of sequence divergence across the genus [24–27], one of which used ITS sequences to infer species relationships . These data revealed the presence of allopolyploids and complex patterns of interspecific hybridisation at the diploid level. Cytogenetic studies in Nicotiana have revealed that 35S rDNA can occupy as many as five loci on different chromosomes [17, 28, 29] and in some species of section Tomentosae sub-regions of the rDNA array may also be dispersed . Rapid amplification of novel 35S rDNA units has been observed in the fourth generation descendents of synthetic allopolyploids, artificially created to resemble natural N. tabacum. Indeed the natural allotetraploid N. tabacum, thought to have formed <200 000 years ago from relatives of modern N. sylvestris and N. tomentosiformis[32, 33], shows signatures of rapid concerted evolution, where most of the progenitor 35S rDNA units have been replaced by a variant that most closely resembles the N. tomentosiformis unit type [34, 35].
In this work, we addressed inter- and intragenomic homogeneity of rDNA arrays in four diploid Nicotiana species using two platforms of next generation sequencing (Roche 454 and Illumina) coupled with classical Sanger method. We sequenced amplicons covering equal portions of coding (18S gene) and non-coding (ITS1) regions (Figure 1). With these data, we compared the families and levels of divergence of units in both regions and interpreted the data in relation to the numbers of rDNA loci and levels of divergence in the intergenic spacer sequences (IGS). We found evidence for near-complete homogeneity of coding sequence irrespective of locus and copy-number. The non-coding region shows significantly higher divergence within those species that harbour multiple rDNA loci.
The following accessions were used: i) Nicotiana sylvestris Speg. & Comes ac. ITB626 originating from the Tobacco Institute, Imperial Tobacco Group, Bergerac, France. ii) Nicotiana tomentosiformis Goodsp. ac. NIC 479/84 (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany). iii) Nicotiana otophora Griesebach ac. 406/76 (Royal Botanic Gardens, Kew, UK) and iv) N. kawakamii Y. Ohashi (voucher FN568429, Natural History Museum, London, UK).
Preparation of sequencing amplicons by emulsion PCR
Emulsion PCR was used in all steps in order to prevent formation of chimeric DNA during amplification. We first separated individual rDNA units in genomic DNA by Hin dIII restriction enzyme digestion, for which there is at least one restriction site distal to the 18S-5.8S region . About 15 μg of genomic DNAs from N. sylvestris, N. tomentosiformis, N. kawakamii and N. otophora was digested using an excess of enzyme (5 U μg-1 DNA, twice for 8 h). Digested DNAs were precipitated with isopropanol, washed with 70% ethanol and re-dissolved in TE to concentrations of about 150–200 ng μl-1.
For the emulsion PCR we essentially followed the protocol of Williams et al. . Oil-surfactant mixture was prepared by thorough mixing of Span 80 (225 μl; SIGMA, USA), Tween 80 (20 μl; DIFCO, UK), Triton X-100 (2.5 μl; SIGMA, USA) and mineral oil (to 5 ml; SIGMA, USA) in a 15-ml centrifuge tube at 25°C. Four hundred μl of the oil-surfactant mixture was transferred into a CryoTube vial and stirred until use.
The composition of the aqueous phase (230 μl) for the emulsion was as follow: 1x PfuUltra II reaction buffer, 10 g l-1 BSA, 300 nM each forward and reverse primer, 200 μM each dNTP, 5 ng μl-1 of template DNA and 4.6 μl of PfuUltra II Fussion HS DNA Polymerase (Stratagene, USA). The amount of genomic DNA was reduced to a minimum in order to maintain an excess of aqueous droplets over template molecules. The tripartite structure of primers was as follows: 454 sequencing adaptors (italics), variable hexanucleotide TAG sequence (normal letter) and a gene specific 3’ end (bold). Primer A (5’-CGTATCGCCTCCCTCGCGCCATCAG TCGTATCTACACTGATGTATTCAACGAG-3’) contained the conserved 18S gene; Primer B (5’-CTATGCGCCTTGCCAGCCCGCTCAG TCGTATCCGTTGCCGAGAGTCGTTT 3’) was derived from the 5.8S gene (Figure 1). The gene-specific parts are identical to 18S-FOR and 5.8S-REV2 primers used in previous projects  and do not seem to discriminate between individual alleles. Two hundred μl of the aqueous phase was added in a drop-wise manner to the 400 μl of stirred oil-surfactant mixture for over a period of 1.5-2.0 min. Stirring was continued for an additional 5 min and then the whitish emulsion was divided into 50 μl aliquots. Approximately 20 μl of aqueous phase was used as a control. The samples were overlaid with a drop of mineral oil and subjected to temperature-cycling: initial denaturation at 94°C for 3 min was followed by 25 cycles of 94°C for 30 s, 57°C for 30 s and 72°C for 40 s. The emulsified PCR reactions were pooled into a 1.5 ml microcentrifuge tube and centrifuged at 13,000g for 5 min at room temperature. The upper (oil) phase was removed. In order to break the emulsion, 2–3 extractions with water-saturated diethyl ether were performed (approximately 1 ml of ether was added, vortexed and the upper phase removed). Residual ether was removed by incubation 15 min at 42°C or by a centrifugation under vacuum for 5 min at room temperature. The PCR products were checked by agarose gel electrophoresis and cleaned using a PCR purification kit (Macherey-Nagel, Germany). The binding buffer was adjusted to remove all unincorporated primers according to the manufacturer’s recommendation.
Samples containing 100–200 ng of each tagged PCR product were pooled and sent to EuroFins Operon MWG (Germany) for 454 sequencing. The pooled sample was sequenced from Primer A- and Primer B sides using a Roche GS FLX apparatus and Titanium series chemistry. Average length of reads was 350 to 450 base pairs and these reads are available at the NCBI Sequence Read Archive under the accession number SRA051350.
Amplicon variance analysis was carried out at Eurofins MWG Operon (Germany) and included Mira coverage mapping and syntheny plots. The SNPs were detected in the 454SNP/454HCDiff.files retrieved from the GsMapper. The criteria for clustering were as follows: All alignments were performed using the Smith-Waterman algorithm with default parameters with an identity threshold of e-100 and a maximum of 10 mutations per read. The reference sequences used for mapping were obtained from direct Sanger sequencing of 18S-5.8S PCR products. In general, half of the reads were successfully mapped to the reference sequences. The unmapped reads contained mostly shorter sequences or unrelated and highly variable sequences that were excluded from further analysis. The lengths of reference sequences were as follows: 313 bp of the 18S (all species), 240±2 bp of the ITS1. The 313 bp of 18S gene is delimited by positions 1689–1801 of the 18S gene from tomato (X51576.1); positions of ITS1 were according to the sequences deposited in GenBank (AJ492423.1, AJ492450.1, AJ492455.1, AJ492445.1, ). The sequences from major (>5% of total reads) clusters (equivalent to gene families) were imported to the BIOEDIT sequence editor  and checked by eye.
Illumina genomic DNA sequencing
Sequence reads were obtained from Illumina sequencing of N. sylvestris (ac. ITB626) and N. tomentosiformis (ac. NIC 479/84) genomic DNA using the Illumina Genome Analyzer xII at the Genome Centre Queen Mary University of London, as described in Renny-Byfield et al. . A random sample between 47-61% of the genome was sequenced for each species. Sequence reads are available at the SRA under the study accession number SRA045794.1. Illumina reads were mapped to ITS reference sequences and SNP and DIP (insertion/deletion) analysis was carried out using GLC Genomics Workbench with the following parameters/requirements: window length of 11 bp, maximum of 2 gaps, a minimum coverage of 4, variants should occur at a minimum frequency of 0.01, with a maximum of 1000 variants expected.
Analysis of clones
ITS sequences were obtained by PCR amplification of genomic DNA. In a 25 μl reaction we used 0.1–1 ng genomic DNA as template, 500 nM each primer, 200 μM each dNTP, and 0.4 units of DyNAzyme II DNA polymerase (Finnzymes, Espoo, Finland). Cycling conditions were as follows: an initial denaturation step of 94°C for 180 s followed by 15–35 cycles of 94°C for 20 s, 57°C for 30 s and 72°C for 30 s with 18S-FOR (5’-GCGCTACACTGATGTATTCAACGAG-3’) and 5.8S-REV (5’-CGCAACTTGCGTTCAAAGACTCGA-3’) primers . The products were purified using a PCR purification kit (Macherey-Nagel, Germany) and cloned into a dT vector (pDrive, Qiagen, Germany). Sequencing was carried out by dideoxy chain-termination at the Eurofins MWG Operon (Germany) using the SP6 and T7 primers. We considered the possibility of differential amplification of GC-rich and GC-poor templates [39, 40] and included 5% dimethyl sulfoxide in the PCR reaction and found this had no effect on the proportion of amplified gene families.
Southern blot hybridisation
DNA was extracted from fresh young leaves according to Kovarik et al. , digested with Eco RV restriction endonuclease (5 U μg-1 DNA, twice for 6 h), fractionated by gel electrophoresis and transferred to Hybond XL membranes (GE-Healthcare, UK) using alkaline capillary transfer. Membranes were hybridised with 32P-labelled DNA probe (DecaLabel DNA Labeling Kit, MBI Fermentas). Southern blot hybridisation was carried out in a 0.25 M sodium phosphate buffer (pH 7.0) supplemented with 7% (w/v) sodium dodecyl sulphate (SDS) at 65°C. Membranes were washed with 2x SSC, 0.1% SDS (twice for 5 min) and then with 0.2x SSC and 0.1% SDS (twice for 15 min at 65°C). The membranes were exposed to a Storage Phosphor Screen, scanned (Storm, GE-Healthcare, USA) and the signal was quantified using Image Quant (GE-Healthcare, USA). The DNA probe was an insert of the clone carrying the 18S gene sequence from tomato (GeneBank number X51576).
Summary of 454 sequencing data
Number of loci1
Number of clusters
Comparing data between sequencing methods
To validate the results we compared frequencies of SNPs obtained with different sequencing methods across N. sylvestris ITS1 sequences (Additional file 7). We cloned 18S-ITS1 PCR products from N. sylvestris and sequenced (15 clones) by Sanger method. As expected, we did not observe any variation in the 18S region, confirming high levels of homogeneity. However, three clones (20%) contained a C➝T substitution at position 57 in ITS1. These clones were identical to the 454 reads in the second most abundant cluster (Figure 2, yellow field). Likewise SNP analysis of Illumina datasets revealed the same substitution, occurring at a frequency of 15.5% (Figure 3). Thus all three methods are broadly comparable.
Among the 3882 N. sylvestris ITS sequences derived from 454 data, there were 680 reads with indels and 800 reads harbouring substitutions (1.2 substitution/indel ratio). The ratio was significantly higher (18.1 substitution/indel ratio) among the Illumina reads indicating that most indels in the 454 data are likely to be artefacts. Indeed, visual inspection of sequences showed that most indels were actually located in homopolymeric tracts, where pyrosequencing is known to introduce errors . Illumina seems to be less prone to indel artefacts, although even in this case, one abundant indel variant (15% reads) could not be traced among the 454 reads or Sanger sequences. We recommend caution when interpreting indel polymorphisms from such NGS data, and suggest using a combination of approaches.
Phylogenetic relationships between the ITS1 families
Southern blot hybridisation
To study concerted evolution of rDNA arrays during the divergence of diploid Nicotiana species, we analysed SNPs in the 18S (coding) and ITS1 (non-coding) regions using Roche 454 and Illumina technology. The coverage of both 18S (3’ end) and ITS1 regions of rDNA was close to 1, meaning that most rDNA variants are likely to be represented in our dataset. We show that 18S sequences are more homogenous than ITS1; a pattern that could arise from incomplete rounds of homogenisation and stronger purifying selection acting on the 18S genic region. By analysing genomic and cytogenetic data, we find evidence that, in Nicotiana, the separation of rDNA arrays between chromosomes influences patterns of rDNA homogenisation. We explore these ideas in more detail below.
Homogeneity in coding and non-coding regions of the rDNA unit
Nicotiana tomentosiformis has only one read rich-cluster (defined as a cluster comprising more than 5% of the reads) for both 18S and ITS1 sequences (Figure 2). These data demonstrate near complete homogenisation of both 18S and ITS1 sequences to a single ribotype. For the other diploid species N. sylvestris, N. kawakamii and N. otophora there was also a single read rich 18S rDNA cluster, but there were two or three read rich ITS1 clusters that differed by 1–3 substitutions per 240 bp (0.4-1.3% divergence; Figure 2). Thus, within each of these species the 18S gene is more homogenous than the ITS1. The average number of major ITS1 variants in Nicotiana is similar to the average number (2–3) of ITS2 ribotypes recently determined for a wide range of plant species by pyrosequencing  corroborating the hypothesis that amplification and fixation of rDNA variants could be larger in plants than in yeasts  and Drosophila. Consistent with previous studies [48, 49], for IGS sequences there is even greater complexity in the Nicotiana species analysed (Figure 5) probably due to its sub-repeated nature ( and Additional file 8). Increasing genetic variation, 18S < ITS1 < IGS likely reflect reducing strengths of selection. Probably the IGS homogenises by similar mechanisms (e.g. unequal cross over) as the rest of the unit since the first and last A1/A2-subrepeats (Figure 1) are more variable than those of the central region , consistent with distance-dependent models of repeat evolution [51, 52].
Most rDNA ribotypes have not survived species divergence in Nicotiana
Chromosomal locus separation as a driver of ITS complexity
Large scale analysis of rDNA repeats in yeasts  and Drosophila has also revealed differences among rDNA units of the same species. However, unlike the pattern we report here, intragenomic homogeneity is similar in coding and non-coding regions of each species, although slightly higher variation was observed in Drosophila, where rDNA sequences are located at several chromosomal loci. In explaining the level of variation, it is suggestive that the Rosaceae [60, 61] and Cactaceae , in which large numbers of ITS variants were observed, also have a high (>5) number of rDNA loci [62, 63]. A comparable pattern is reported within the grasshopper species Podisma pedestris, in which some populations have a larger number of rDNA loci and a greater complexity of rDNA units . A similar correlation between locus number (Figure 6) and ITS1 complexity (Figure 2) emerges from our results. For example, N. tomentosiformis, with a single locus, has highly homogeneous 18S and ITS1 sequences, resembling the situation in unicellular yeast. On the other hand, N. kawakamii with four loci had the highest variability amongst ITS sequences.
Low copy numbers of rDNA units (or parts of the unit) can be found dispersed across the Arabidopsis  and some animal [71, 72] genomes. IGS dispersion may not be uncommon in plant genomes since it has also been reported in other species, and proposed to be one of the mechanisms leading to formation of novel high copy satellites [73–75]. Indeed, we have observed this phenomenon in Nicotiana section Tomentosae, where there is dispersion of IGS sequences and 35S rDNA across the genome . Such units could potentially nucleate the expansion of active rDNA loci. Interestingly, N. kawakamii, with the highest level of rDNA dispersion (Figure 6), also has the most heterogeneous ITS1 ribotypes.
New loci and units have been shown to evolve rapidly in early generations of synthetic allopolyploids [31, 76–79] or in response to DNA damage . In an allotetraploid line of tobacco, we observed amplification of new IGS types at a new rDNA locus .
Transposable elements may be vehicles for rDNA mobility and the generation of orphaned rDNA units across the genome. For example in wheat mobile rDNA sites may be connected with meiotic activity of En/Spm elements , while Ty1/Copia elements have been found in the IGS of Allium cernuum .
The results here indicate that ITS1 is more heterogeneous than 18S, particularly when more than one rDNA locus is present. Low homogenisation frequency between rDNA arrays at different loci, together with strong selection pressures imposed on coding regions, would allow co-existence of repeats with identical coding but more variable non-coding regions. We suggest that locus duplication or amplification of orphaned rDNA units can be vehicles for rDNA divergence (Figure 7).
We thank Jana Kubíčková for help with sorting the SNP analysis data. The work was supported by the Czech Science Foundation (P501/10/0208; P501/12/G090; 206/09/1751), Academy of Science of the Czech Republic (RVO68081707), the Czech-French bilateral Barrande program (09150SH), Natural Environment Research Council, UK, and Agence Nationale de la Recherche (ANR) research contract (Association pour la Recherche sur les Nicotianées).
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