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

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.