Structure and Methylation of 35S rDNA in Allopolyploids Anemone multifida (2 n = 4 x = 32, BBDD) and Anemone baldensis (2 n = 6 x = 48, AABBDD) and Their Parental Species Show Evidence of Nucleolar Dominance

Jelena Mlinarec; Ljudevit Luka Boštjančić; Nenad Malenica; Adela Jurković; Todd Boland; Sonja Siljak Yakovlev; Višnja Besendorfer

doi:10.3389/fpls.2022.908218

Structure and Methylation of 35S rDNA in Allopolyploids Anemone multifida (2 n = 4 x = 32, BBDD) and Anemone baldensis (2 n = 6 x = 48, AABBDD) and Their Parental Species Show Evidence of Nucleolar Dominance

Front Plant Sci. 2022 Jul 6:13:908218. doi: 10.3389/fpls.2022.908218. eCollection 2022.

Authors

Jelena Mlinarec¹, Ljudevit Luka Boštjančić^{2

3}, Nenad Malenica⁴, Adela Jurković⁴, Todd Boland⁵, Sonja Siljak Yakovlev⁶, Višnja Besendorfer⁴

Affiliations

¹ Oikon ltd.-Institute of Applied Ecology, Zagreb, Croatia.
² LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), Senckenberg Biodiversity and Climate Research Centre, Senckenberg Gesellschaft für Naturforschung, Frankfurt, Germany.
³ Department of Computer Science, ICube, UMR 7357, CNRS, Centre de Recherche en Biomédecine de Strasbourg, University of Strasbourg, Strasbourg, France.
⁴ Division of Molecular Biology, Department of Biology, University of Zagreb, Horvatovac, Croatia.
⁵ Memorial University of Newfoundland's Botanical Gardens, St. John's, NL, Canada.
⁶ CNRS, AgroParisTech, Ecologie Systématique Evolution, Université Paris-Saclay, Orsay, France.

Abstract

Transcriptional silencing of 35S rDNA loci inherited from one parental species is occurring relatively frequently in allopolyploids. However, molecular mechanisms by which it is selected for transcriptional silencing remain unclear. We applied NGS, silver staining and bisulfite sequencing to study the structure, expression and methylation landscape of 35S rDNA in two allopolyploids of common origin, allotetraploid Anemone multifida (2n = 4x = 32, genome composition BBDD) and allohexaploid A. baldensis (2n = 6x = 48, AABBDD), and their genome donors, A. sylvestris (2n = 16, AA), A. cylindrica (2n = 16, BB) and A. parviflora (2n = 16, DD). The size of the recovered 35S rDNA units varied from 10,489 bp in A. cylindrica to 12,084 bp in A. sylvestris. Anemone showed an organization typical of most ribosomal 35S rDNA composed of NTS, ETS, rRNA genes, TTS and TIS with structural features of plant IGS sequences and all functional elements needed for rRNA gene activity. The NTS was more variable than the ETS and consisted of SRs which are highly variable among Anemone. Five to six CpG-rich islands were found within the ETS. CpG island located adjacent to the transcription initiation site (TIS) was highly variable regarding the sequence size and methylation level and exhibited in most of the species lower levels of methylation than CpG islands located adjacent to the 18S rRNA gene. Our results uncover hypomethylation of A. sylvestris- and A. parviflora-derived 35S rDNA units in allopolyploids A. multifida and A. baldensis. Hypomethylation of A. parviflora-derived 35S rDNA was more prominent in A. baldensis than in A. multifida. We showed that A. baldensis underwent coupled A. sylvestris-derived 35S rDNA array expansion and A. parviflora-derived 35S rDNA copy number decrease that was accompanied by lower methylation level of A. sylvestris-derived 35S rDNA units in comparison to A. parviflora-derived 35S rDNA units. These observations suggest that in A. baldensis nucleolar dominance is directed toward A. sylvestris-derived chromosomes. This work broadens our current knowledge of the 35S rDNA organization in Anemone and provides evidence of the progenitor-specific 35S rDNA methylation in nucleolar dominance.

Keywords: CpG islands; IGS; Ranunculaceae; bisulfite sequencing; epigenetic modification; genome size; polyploidy; silver staining.