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Concerted evolution rapidly eliminates sequence variation in rDNA coding regions but not in intergenic spacers in Nicotiana tabacum allotetraploid

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Abstract

Nicotiana tabacum (tobacco) is a natural allotetraploid that formed from two diploid progenitors (N. sylvestris—S-genome, N. tomentosiformis—T-genome) within past 0.2 million years. Previous classical studies have shown that its 35S rDNA has been largely homogenised towards T-genome-like homeologs. However, the degree of conversion at single nucleotide resolution remains unknown. Here, we analysed intragenomic variation of rDNA at high resolution in natural tobacco, synthetic tobacco and the progenitors employing genomic, molecular and cytogenetic methods. In synthetic tobacco, we identified 13 highly (≥10% units) polymorphic sites in the 18S-5.8S-26S coding region. In contrast, only a single polymorphic site was detected in natural tobacco, indicating that gene conversion has removed most of the polymorphisms over shallow evolutionary times. However, the non-coding 26S-18S intergenic spacer (IGS) was highly polymorphic in both natural (57 polymorphic sites) and synthetic tobacco (128 polymorphic sites). In natural tobacco, most (64%) IGS polymorphisms were inherited from the N. tomentosiformis progenitor, while 36% appeared de novo indicating rapid rates of sequence divergence of IGS. FISH revealed that the T-genome-like units (harbouring N. tomentosiformis-type IGS) occurred on all four loci in tobacco variety 095-55, including those loci derived from N. sylvestris progenitor, while the variety SR-1 retained 1–2 S-genome loci unconverted and transcriptionally silenced. We discuss potential caveats associated with experimental and in silico approaches used for determination of rDNA polymorphisms. We also hypothesise that polyploidy-associated gene conversion may eliminate mutated and non-functional genes that have accumulated in progenitor genomes, thereby contributing to success of polyploidy species.

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Acknowledgements

The research was funded by the Czech Science Foundation (P501/17/11642S and P506/16-02149J) and NERC.

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Authors and Affiliations

  1. Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., 61265, Brno, Czech Republic

    J. Lunerová, R. Matyášek & A. Kovařík

  2. School of Biological and Chemical Sciences, Queen Mary University of London, London, UK

    S. Renny-Byfield & A. Leitch

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  1. J. Lunerová
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  2. S. Renny-Byfield
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  3. R. Matyášek
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  4. A. Leitch
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  5. A. Kovařík
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Correspondence to A. Kovařík.

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Handling Editor: Miloslav Plohl.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1.

Sequence assembly of full length rDNA units from the GenBank clones. These sequences were used as references to build NGS consensus. (PDF 174 kb)

Online Resource 2.

Sequence alignments of Nicotiana 35S rDNA (18S-ITS1-5.8S-ITS2-26S-IGS) reference and NGS consensus sequences. (a) A low resolution view. Coding regions and promoters are annotated with brown and red arrows, respectively. Regions of probes hybridisation are annotated with green arrows. Reference sequences used for haplotypic analysis and cDNA reads map** are in cyan blue. PCR amplicons used in RT-CAPS analysis (Fig. 5) are in violet. Polymorphic restriction sites are in dark blue. (b) Nucleotide level-resolution view. (PDF 9768 kb)

Online Resource 3.

Data sets. Sheet 1 – List of DNA polymorphisms in N. sylvestris units. The polymorphic sites included single-nucleotide substitutions (SNV), insertions (INS), deletions (DEL) and multi-nucleotide variations (MNV). Sheet 2 – List of DNA polymorphisms in N. tomentosiformis units, as in sheet 1. Sheet 3 - List of DNA polymorphisms in N. tabacum var. SR-1 units, as in sheet 1. Sheet 4 - List of DNA polymorphisms in synthetic tobacco line TR1-A, as in sheet 1. Sheet 5 - List of DNA polymorphisms in an electronic N. sylvestris and N. tomentosiformis hybrid, as in sheet 1. Sheet 6 – Pairwise comparisons of NGS consensus and GenBank clones. Sheet 7 – Lengths of rDNA units and subregions. Sheet 8 – Summary of SNPs in the IGS region. Sheet 9 - Haplotypic analysis of rDNA subregions. Sheet 10 – Comparison of rDNA alleles between N. tabacum and N. tomentosiformis. Sheet 11 – ITS1 amplicon sequencing – cDNA. Sheet 12 – ITS1 amplicon sequencing – gDNA. Sheet 13 – Summary of ITS1 amplicon sequencing. (XLSX 187 kb)

Online Resource 4.

Alignment of ITS1 consensus sequences from N. sylvestris (SYL58) and N. tomentosiformis (TOM58). (PDF 362 kb)

Online Resource 5.

Analysis of tandem repeats in the A-subregion of IGS in N. tabacum, N. tomentosiformis, N. sylvestris. (PDF 401 kb)

Online Resource 6.

Positions of restriction endonuclease recognition sites and regions of 26S probe hybridisation (rectangle) in rDNA units. (PDF 536 kb)

Information on Electronic Supplementary Material

Information on Electronic Supplementary Material

Online Resource 1. Sequence assembly of full length rDNA units from the GenBank clones. These sequences were used as references to build NGS consensus.

Online Resource 2. Sequence alignments of Nicotiana 35S rDNA (18S-ITS1-5.8S-ITS2-26S-IGS) reference and NGS consensus sequences. (a) A low resolution view. Coding regions and promoters are annotated with brown and red arrows, respectively. Regions of probes hybridisation are annotated with green arrows. Reference sequences used for haplotypic analysis and cDNA reads map** are in cyan blue. PCR amplicons used in RT-CAPS analysis (Fig. 5) are in violet. Polymorphic restriction sites are in dark blue. (b) Nucleotide level-resolution view.

Online Resource 3. Data sets. Sheet 1 – List of DNA polymorphisms in Nicotiana sylvestris units. The polymorphic sites included single-nucleotide substitutions (SNV), insertions (INS), deletions (DEL) and multi-nucleotide variations (MNV). Sheet 2 – List of DNA polymorphisms in N. tomentosiformis units, as in sheet 1. Sheet 3 - List of DNA polymorphisms in N. tabacum var. SR-1 units, as in sheet 1. Sheet 4 - List of DNA polymorphisms in synthetic tobacco line TR1-A, as in sheet 1. Sheet 5 - List of DNA polymorphisms in an electronic N. sylvestris and N. tomentosiformis hybrid, as in sheet 1. Sheet 6 – Pairwise comparisons of NGS consensus and GenBank clones. Sheet 7 – Lengths of rDNA units and subregions. Sheet 8 – Summary of SNPs in the IGS region. Sheet 9 - Haplotypic analysis of rDNA subregions. Sheet 10 – Comparison of rDNA alleles between N. tabacum and N. tomentosiformis. Sheet 11 – ITS1 amplicon sequencing – cDNA. Sheet 12 – ITS1 amplicon sequencing – gDNA. Sheet 13 – Summary of ITS1 amplicon sequencing.

Online Resource 4. Alignment of ITS1 consensus sequences from Nicotiana sylvestris (SYL58) and N. tomentosiformis (TOM58).

Online Resource 5. Analysis of tandem repeats in the A-subregion of IGS in Nicotiana tabacum, N. tomentosiformis, N. sylvestris.

Online Resource 6. Positions of restriction endonuclease recognition sites and regions of 26S probe hybridisation (rectangle) in rDNA units.

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Lunerová, J., Renny-Byfield, S., Matyášek, R. et al. Concerted evolution rapidly eliminates sequence variation in rDNA coding regions but not in intergenic spacers in Nicotiana tabacum allotetraploid. Plant Syst Evol 303, 1043–1060 (2017). https://doi.org/10.1007/s00606-017-1442-7

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  • DOI: https://doi.org/10.1007/s00606-017-1442-7

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