Ribosomal DNA rDNA rRNA Organization and Evolution at Large Scale IBC2024

5S and 45S rDNA monomer organization: lengths, variation and interruption in tandem arrays from Musaceae species
Authors
Pat Heslop-Harrison1,2, Qing Liu2 , Ziwei Wang2, Trude Schwarzacher1,2
Affiliations
1 University of Leicester, Leicester, United Kingdom 2 South China Botanical Garden, Guangzhou, China
Abstract
Long, single-molecule DNA sequencing shows the organization and structures of rDNA monomers in tandem repeats. Short reads of both 5S and 45S rDNA collapse the arrays during assembly, while older BAC sequences suffer from chaemerism and assembly artefacts. Far from being a continuous array of monomers, we find short deletions, insertions or interruptions in the arrays. Full-length retroelements are found at variable points within some 45S and 5S monomers in the arrays, and there are occasional insertions of uncharacterized sequences. Within monomers, both deletions and short duplications are found. Similar rearrangements have been found in multiple, non-identical, reads, giving evidence for homogenization through unequal crossing-over (and hence duplication of segments of the arrays). The 'starts' of the arrays have been characterized with flanking sequences. Musaceae provides a good model for the comparative study of the rDNA arrays, with long reads available from multiple species, variable chromosome numbers and evolutionary movement of rDNA between chromosomes, independent of other genes. The rDNA is very variable between species, many with one pair sites of 45S rDNA, representing 1% of all the DNA, to Musa beccarii with 3 sites and 5% of the DNA. Monomer lengths are also variable, with the typical length around 400bp found for most 5S monomers but 1056bp in Ensete. The detailed characterization of the arrays shows evolutionary mechanisms and diversity of the ribosomal DNA arrays. Further information and references are given at www.molcyt.org .
IBC2024 Madrid International Botanical Congress XX XXIBC

1. 5S and 45S rDNA monomer organization: lengths, variation and interruption in tandem arrays from Musaceae species Evolutionary mechanisms driving genome diversification Pat Heslop-Harrison Twitter: @PatHH1 www.molcyt.org [email protected] With Qing Liu, Mathieu Rouard, Ziwei Wang and Trude Schwarzacher University of Leicester, Leicester, United Kingdom South China Botanical Garden, Guangzhou, China

2. Dessert banana Musa 2n=3x=33; AAA genomes Musa assembly: d’Hont , HH et al. Ensete : Wang, HH et al 2022 Musa beccarii Wang, XueYun Ge 2023 Musella lasiocarpa Wang et al. 2025 Banana Genome Hub Droc et al. 2022 DAPI chromosomes 5S rDNA 45S rDNA www.molcyt.org Pat Heslop-Harrison [email protected]

3. Ribosomal RNA Illumina unselected RNA sequencing RNA: precursor to life - catalytic and self-assembling Ribosomal RNA comes next Ribosomes are able to make any other protein from RNA ( and then came reverse transcriptase – retrotransposons and telomerase) rRNA is universal and an abundant transcript Zhou S, Richert-Pöggeler KR, Wang Z, Schwarzacher T, Heslop-Harrison JS, Liu Q. 2022.  High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental  Hibiscus .   Acta Horticulturae  1392  10.17660/ActaHortic.2024.1392.4   bioRxiv 2022.01.25.477650 & in preparation Species rDNA Chloroplast Mitochondrion Remaining reads Contigs reads rate reads rate reads rate reads rate Brassica rapa 42,807,438 48.2% 30,329,502 34.1% 1,358,581 1.5% 13,726,350 15.4% 63,963 Carica papaya 59,996,491 80.6% 2,959,838 3.98% 1,741,383 2.34% 8,895,916 12.0% 72,184 Lycium barbarum 50,975,986 51.4% 27,539,934 27.8% 462,012 0.5% 19,322,488 19.5% 41,325

4. Evolution of rDNA – genes and spacers 18S 5.8S ITS2 ITS1 NTSL 26S MuTR NTSR 45S rDNA repeat unit: typically 10kb monomer www.molcyt.org Pat Heslop-Harrison [email protected] 5S rDNA repeat unit: typically 450bp monomer 5S IGS ITS internal transcribed spacer IGS inter gene spacer NTS non-transcribed spacer L/R Left/Right MuTR tandem repeat (Musaceae) S Svedberg unit – size of the transcribed molecule from density gradient centrifugation of RNA

5. Genome sequencing and repeats BAC sequencing: assembly algorithms of Sanger reads +/- failed put variants together (in 2010 couldn’t publish nor even store each read/trace) Illumina: very high accuracy (Q>40) not a hope to reconstruct from 150bp paired-end reads PacBio: high accuracy (2022 Q>20) but typically 15kb reads, only 1.5 45S rDNA monomers so no hope to understand array organization Nanopore: low accuracy (Q<8) but many >100kb Repetitive DNA discovery www.molcyt.org Pat Heslop-Harrison [email protected]

6. Can we assemble the DNA sequence in repeats? Sequencing depth graph Individual reads overlap Short reads assemblies ‘collapse’, with all reads ‘on top’ of each other – Not variable enough to assemble or not enough overlap www.molcyt.org Pat Heslop-Harrison [email protected]

7. 0.078% of the genome or ∼ 366 copies, with a complete motif length of 1,056 bp. first 400 bp of the intergenic spacer had no significant BLAST hits in GenBank; second part showed short regions with weak homology Musa chromosome assemblies ± All copies are the long monomer – homogenization Why is the monomer length for the 5S rDNA in E. glaucum so long? 5S rDNA repeat unit: typical 450bp monomer (Musaceae & all spp ) ITS Ensete glaucum 5S rDNA repeat unit: 1056bp monomer 5S 119bp ITS 937bp Ziwei Wang, HH, Qing Liu. 2022.  A chromosome-level reference genome of  Ensete glaucum  gives insight into diversity, chromosomal and repetitive sequence evolution in the Musaceae.  Gigascience   11:  1-21. 5S 119bp

8. Evolution of rDNA – genes and spacers Sequence and length of monomer (PacBio > yr 2022) Number of loci of arrays ( in situ hybridization FISH & high quality assemblies) Organization of arrays on chromosomes (FISH & assembly) Number of monomers in array (FISH; total in cell by Illumina reads; 2024 struggling with assembly) Evolution and variation of monomers in arrays 18S 5.8S ITS2 ITS1 NTSL 26S MuTR NTSR 45S rDNA repeat unit: typically 10kb monomer www.molcyt.org Pat Heslop-Harrison [email protected] 5S rDNA repeat unit: typically 500bp monomer 5S ITS

9. F Cen FAM 5S Cy3 5S rDNA monomers Nanopore read (0-30,000bp) self dotplot EGL05 eg05 Wang, HH, Qing Liu. 2022.  A chromosome-level reference genome of  Ensete glaucum  gives insight into diversity, chromosomal and repetitive sequence evolution in the Musaceae.  Gigascience   11:  1-21. EGL05 multiple 5S rDNA hybridization sites several, non-continuous, signals visible non-continuous nature of the 5S rDNA array EGL05

10. Nanopore read (0-30,000bp) self dotplot 1kb 5S rDNA tandem repeat from Ensete glaucum www.molcyt.org Pat Heslop-Harrison [email protected]

11. F 45S FITC Cen Cy3 X 45S green  5S Cy3 Cen FAM 5S Cy3 ONT read (3,000-14,500nt) retroelement LTR LTR 5S rDNA monomers 5S rDNA monomers 5S rDNA monomers 5S rDNA monomers ONT read (1,000-24,000nt) Mb eg05 eg05 eg05 eg06 eg06 eg05 eg06 eg06

12. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR ZiWei Wang, Qing Liu 45S rDNA repeat unit in Musella lasiocarpa and Ensete glaucum Oh dear! – Impossible to assemble! Nanopore read accuracy Q=8, 87.5% Illumina 150bp; PacBio 15kb

13. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR ZiWei Wang, Qing Liu

14. Evolution involves Insertion of an LTR retroelement within the 45S rDNA array

15. Sometimes: Not a well-known retroelement

16. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR 18S ITS1 MuTR NTSR www.molcyt.org Pat Heslop-Harrison [email protected] Deletion of part of monomer and small duplication

17. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR 18S ITS1 MuTR NTSR www.molcyt.org Pat Heslop-Harrison [email protected]

18. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR 18S ITS1 MuTR NTSR www.molcyt.org Pat Heslop-Harrison [email protected] Evolution involves mutation (deletion/duplication) then 2) copying of deleted monomer alternatively reiteration of the same deletion/duplication event

19. 5S rDNA: head-to-head junction in array of monomers

20. 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR 41123-3de2 NTSL 26S STR MuTR NTSR 18S 5.8S ITS2 ITS1 NTSL 26S STR MuTR NTSR www.molcyt.org Pat Heslop-Harrison [email protected]

21. Unlikely but possible: Nanopore sequencing: hypothetical artifact of head-to-head junction

22. Self-annealing ‘zipping up’ Second strand continues though pore

23. 5S and 45S rDNA monomer organization: lengths, variation and interruption in tandem arrays from Musaceae species Evolutionary mechanisms driving genome diversification rDNA shows within and between species variation Moving between chromosomes independently of genes Evolving and homogenizing in each species Deletions and rearrangements retroelements and other insertions Array variation Pat Heslop-Harrison [email protected] With Qing Liu, Mathieu Rouard, Ziwei Wang and Trude Schwarzacher University of Leicester, Leicester, United Kingdom South China Botanical Garden, Guangzhou, China A Journal to Shape the Future and Understand the Earth

24. Biological Diversity : A Journal to Shape the Future and Understand the Earth Scan to know more about BD All scholars are welcome to submit! Biological Diversity is an international, peer-reviewed, open access, journal ， Hosted by South China Botanical Garden , Chinese Academy of Sciences ， published in cooperation with Wiley . Biodiversity conservation, evaluation and sustainable use of resources Prof. Hai Ren Ecological & Environmental Sciences Center, SCBG,CAS, Guangzhou, China Email ： [email protected] ORCID: 0000-0002-3744-8007 Pat Heslop-Harrison Co-chief editor Cell Biology and Molecular Cytogenetics, University of Leicester, UK Email ： [email protected] ORCID: 0000-0002-3105-2167 Issue 1: March 2024 Editor-in-Chief: [email protected] Editorial office: [email protected] ; [email protected] Article Type: Original article, Review, Report, Commentary, Communication, Perspective, Opinion, Letter, News, Editorial, Methods and Applications