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Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus

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Abstract

The dynamics of genome modification that occurred from the initial hybridization event to the stabilization of allopolyploid species remains largely unexplored. Here, we studied inheritance and expression of rDNA loci in the initial generations of Brassica napus allotetraploids (2n = 38, AACC) resynthesized from Brassica oleracea (2n = 18, CC) and B. rapa (2n = 20, AA) and compared the patterns to natural forms. Starting already from F1 generation, there was a strong uniparental silencing of B. oleracea genes. The epigenetic reprogramming was accompanied with immediate condensation of C-genome nucleolar organizer region (NOR) and progressive transgeneration hypermethylation of polymerase I promoters, mainly at CG sites. No such changes were observed in the A-genome NORs. Locus loss and gains affecting mainly non-NOR loci after the first allotetraploid meiosis did not influence established functional status of NORs. Collectively, epigenetic and genetic modifications in synthetic lines resemble events that accompanied formation of natural allopolyploid species.

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Acknowledgments

Tomasz Książczyk was supported by a fellowship from the Plant Breeding Departement of INRA (France). This work was carried out with the financial support of the “ANR–Agence Nationale de la Recherche–The French National Research Agency” under the “Programme Biodiversite”, project “ANR-05-BDIV-015, Effet de la polyploïdie sur la biodiversité et l’évolution du génome des plantes”. This research was funded by partially funded by the Grant Agency of the Czech Republic (P501-10-0208), the Academy of Sciences of the Czech Republic (AVOZ50040702) and Fr-CZ (Barrande, MEB-020823 and MEB-021114) bi-national Programmes d'Actions Intégrées (PAI). The authors thank Prof. R. Hasterok for providing the 5S and 26S rDNA clones and Dr E. Jenczewski for critical reading of the manuscript.

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  1. Tomasz Książczyk

    Present address: Laboratory of Cytogenetics and Molecular Biology, Institute of Plant Genetics Polish Academy of Sciences, 60-479, Poznań, Poland

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  1. Institut National de la Recherche Agronomique (INRA), UMR118 APBV, BP 35327, 35653, Le Rheu cedex, France

    Tomasz Książczyk, Frédérique Eber, Virginie Huteau, Olivier Coriton & Anne-Marie Chèvre

  2. Institute of Biophysics, Academy of Science of the Czech Republic, Brno, Czech Republic

    Ales Kovarik, Lucie Khaitova & Zuzana Tesarikova

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Tomasz Książczyk and Ales Kovarik contributed equally to this work.

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ESM 1

Scheme showing polymerase I promoter region and amplicons used for bisulfite sequencing (JPEG 3451 kb)

ESM 2

Inheritance of the 35S rDNA units in synthetic lines of Brassica determined by Southern blot hybridization. (a) Map of EcoRI restriction sites within the ITS subregion and the lengths of cleavage products. There is one EcoRI site in the B. oleracea ITS2 while there is none in the ITS2 of B. rapa. Both types of parental units have conserved EcoRI sites in the 18S and 26S genes. b–e Southern blot hybridization of genomic DNA restricted with EcoRI and subsequently probed with the ITS1 probe lines CD, colchicine treatment (b); line UG, unreduced gametes (c); natural B. napus (d); and diploid progenitors (e). Note a strongly enhanced A-genome signal in the S1 individual of line 1UG. The A/C ratios were determined by counting radioactivity of the upper and bottom bands; minor signals in the middle of the gel were not included in the phosphorimaging analysis (JPEG 170 kb)

ESM 3

Bisulfite sequencing analysis. Summarization of methylation frequencies in different sequence motifs (JPEG 2791 kb)

ESM 4

Bisulfite sequencing analysis. Graphs showing distribution of methylated cytosine residues along the sequenced region. Page 1 (ESM 4), B. oleracea genes. Filled and empty symbols represent methylated and non methylated cytosines. The first lanes correspond to master sequences derived from non-treated DNAs. Vertical arrow-transcription start site. A virtually non-methylated clone in the B. oleracea promoter is indicated by an asterisk (JPEG 4913 kb)

ESM 5

Page 2 (ESM 5), B. rapa genes (JPEG 5,225 kb). Filled and empty symbols represent methylated and non methylated cytosines. The first lanes correspond to master sequences derived from non-treated DNAs. Vertical arrow transcription start site. A virtually non-methylated clone in the B. oleracea promoter is indicated by an asterisk (JPEG 5225 kb)

ESM 6

Expression analysis of rDNA in line 1UG by quantitative PCR. Primary rRNA transcript levels (18S-ITS1-5.8S) were determined in root tissues (JPEG 3331 kb)

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Książczyk, T., Kovarik, A., Eber, F. et al. Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus . Chromosoma 120, 557–571 (2011). https://doi.org/10.1007/s00412-011-0331-z

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