Genome sequence assembly of the 5S rDNA loci informs haplotype specificity and evolution in the greater duckweed Spirodela polyrhiza - PubMed (original) (raw)

Genome sequence assembly of the 5S rDNA loci informs haplotype specificity and evolution in the greater duckweed Spirodela polyrhiza

Anton Stepanenko et al. Commun Biol. 2026.

Abstract

Despite the rapid expansion of information on eukaryotic genomes, data on ribosomal DNA (rDNA) loci encoding ribosomal RNAs, crucial for the biogenesis of ribosomes, are absent in almost all cases due to difficulties in assembling the long regions of tandemly repeated DNA units. Taking advantage of the uniquely low rDNA copy number in the aquatic plant Spirodela polyrhiza, we resolved the species' complete 5S rDNA architecture at a nucleotide level. A combination of in situ hybridization, extra-long DNA reads, and conventional DNA sequencing allowed the assembly of near-complete loci sequences of 40,878 bp, specific for one haplotype of chromosome ChrSp6, and of 110,911 bp specific for a haplotype of ChrSp13. The completely resolved 5S rDNA locus of ChrSp6 contains 40 copies of tandemly repeated gene units with an intergenic spacer (NTS) of 400 bp for one haplotype, and more than 60 highly homogenized gene copies for the second haplotype. The ChrSp13 locus contains 5S rDNA clusters with NTSs of 1,056 or 1,069 bp arranged in two sub-clusters. The G/C-rich 5S rDNA arrays in both loci are embedded in A/T-enriched chromosome regions. This work advances our understanding of the basic principles of rDNA organization and evolution of rRNA genes in plants by revealing the molecular architecture and evolutionary dynamics of 5S rDNA loci.

© 2026. The Author(s).

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1

Fig. 1. Molecular and chromosomal variation of 5S rDNA in the S. polyrhiza accession Sp9509.

a PCR fragments amplified with primers specific for 5S rRNA genes; M: DNA size marker, 9509: amplification products from genomic DNA of Sp9509. b Three variants of 5S rDNA repeat units_;_ black arrows represent 5S rDNA primers, red box marks the position of a 13 bp insertion, NTS: non-transcribed intergenic spacer. c FISH signals (left) and volumes after surface rendering of signals in 3D structured illumination microscopy (3D-SIM) image stacks (right) indicate that the amounts of 5SrDNA-Short (red) and 5SrDNA-Long (green) differ between the homologous chromosomes in interphase nuclei (cf. Supplementary Movie 1). * Indicates smaller signals; + indicates larger signals, n = nucleolus.

Fig. 2

Fig. 2. Assembling the 5S rDNA loci on the homologs of ChrSp6 and ChrSp13 of S. polyrhiza Sp9509.

a Schematic representation of the NGS contig and selected representative ONT reads used for assembling sequences composed of short Sp5S-S type 5S rDNA repeats on ChrSp6 (3ab, 154, 9e7c, 185 and fe9 represent corresponding reads 3ab0b3f8-9e42-499a-a733-e7f7ef5a88e3; tig00000154; 9e7ce0b7-2939-4dbb-bae9-bd122f577594; 185db047-daf5-4418-bfc1-3146a719f062 and fe9ebfe0-b4ab-4f96-bb7f-b8aefc7a02d8, listed in Supplementary Table 3). The NGS contig CPO19097.1 is on chromosome #5 of the ASM198140v1 genome, updated to chromosome #6 in the new genome version Sp9509_Oxford_v3, Ns in the NGS contig depiction indicate unresolved repeated DNA units. b Schematic representation of 5S rDNA containing Sp5S-S type gene repeats at homologous chromosomes ChrSp6_A and ChrSp6_B. c Schematic representation of NGS contig and selected representative ONT reads used for assembling sequences composed of long Sp5S-L type 5S rDNA repeats on ChrSp13 (c72, 3d7, 5cf, e8f and e9d correspond to the readsc72d9c80-f704-4d3d-b030-25b9f213fe3c; 3d770410-1945-4622-afd8-7a3257e79119; 5cf44779-0cda-4a54-a602-c253f27d29a6; e8f2492e-3138-4f7f-bf33-7c0c23f3e617 and e9d7a5e2-5a84-4615-a68c-f902b4cce5d4, listed in Supplementary Table 4). The NGS contig CPO19093.1 is on chromosome #1 of the ASM198140v1 genome, updated to chromosome #13 in the new genome version Sp9509_Oxford_v3, Ns in the NGS contig indicate unresolved repeated DNA units. d Schematic representation of 5S rDNA containing Sp5S-L type gene repeats at homologous chromosomes ChrSp13_A and ChrSp13_B. Vertical boxes mark 5S rDNA repeated units, clear boxes in Fig. 2c mark Sp5S-L type 5S rDNA repeats with NTS of 1056 bp (no-13bp insert) and red boxes mark Sp5S-L type 5S rDNA repeats with NTS of 1069 bp (with 13 bp insert); black lines represent raw ONT sequences bordering rDNA clusters; gray horizontal boxes mark consensus border sequences surrounding 5S rDNA clusters; arrows mark the direction of 5S rDNA transcription.

Fig. 3

Fig. 3. G/C plot of the 5S rDNA loci and alignment of border sequences reveal homologous regions on Sp9509 chromosomes.

a G/C plot of the 5S rDNA locus on ChrSp6_B and DNA blast hits of the border elements mapped to the set of all 20 S. polyrhiza chromosomes. b G/C plot of 5S rDNA loci located at ChrSp13_B and DNA blast hits of the border elements are mapped to the set of 20 S. polyrhiza chromosomes.

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