Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus Tomasz Książczyk, Ales Kovarik, Frédérique Eber, Virginie Huteau, Lucie Khaitova, Zuzana Tesarikova, Olivier Coriton & Anne-Marie Chèvre Chromosoma Biology of the Nucleus ISSN 0009-5915 Volume 120 Number 6 Chromosoma (2011) 120:557-571 DOI 10.1007/s00412-011-0331-z
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Author's personal copy Chromosoma (2011) 120:557–571 DOI 10.1007/s00412-011-0331-z
RESEARCH ARTICLE
Immediate unidirectional epigenetic reprogramming of NORs occurs independently of rDNA rearrangements in synthetic and natural forms of a polyploid species Brassica napus Tomasz Książczyk & Ales Kovarik & Frédérique Eber & Virginie Huteau & Lucie Khaitova & Zuzana Tesarikova & Olivier Coriton & Anne-Marie Chèvre
Received: 22 April 2011 / Revised: 23 June 2011 / Accepted: 1 July 2011 / Published online: 23 July 2011 # Springer-Verlag 2011
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.
Communicated by P. Shaw
Introduction
Tomasz Książczyk and Ales Kovarik contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00412-011-0331-z) contains supplementary material, which is available to authorized users. T. Książczyk : F. Eber : V. Huteau : O. Coriton : A.-M. Chèvre (*) Institut National de la Recherche Agronomique (INRA), UMR118 APBV, BP 35327, 35653 Le Rheu cedex, France e-mail:
[email protected] A. Kovarik : L. Khaitova : Z. Tesarikova Institute of Biophysics, Academy of Science of the Czech Republic, Brno, Czech Republic Present Address: T. Książczyk Laboratory of Cytogenetics and Molecular Biology, Institute of Plant Genetics Polish Academy of Sciences, 60-479 Poznań, Poland
Polyploidy, a genome change in which the entire chromosome complement is multiplied (3×, 4×, etc), is particularly widespread in flowering plants, where most species are polyploids or have polyploidy in their genetic history (Soltis et al. 2009; Van de Peer et al. 2009). In plants, polyploidy often occurs in association with interspecific hybridization, a process called allopolyploidy. Most of important crops result from ancient or recent allopolyploidization events (Doyle et al. 2008). The genetic consequences of genome mergers are beginning to be understood mainly from studies of recent natural and synthetic polyploids. Among different systems, one puzzling issue is emerging namely that while some newly formed or resynthesized allopolyploids seem quiescent with few changes in genome organizations such as cotton and Spartina (Baumel et al. 2002), most of the others are extensively remodeled within just few generations
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(Madlung et al. 2005; Skalická et al. 2003; Gaeta et al. 2007; Leitch and Leitch 2008; Szadkowski et al. 2010). In most eukaryotes, nuclear ribosomal 35S rDNA and 5S rDNA units occur in tandem arrays at one or several loci (Hemleben and Zentgraf 1994). Each large 35S rDNA unit contains the 18S, 5.8S, and 26S rDNA genes, the internally transcribed spacers (ITS) and the intergenic spacer. The 5S rDNA encoding for a 120-bp 5S rRNA occur independently from the 35S loci in higher eukaryotes, although their physical linkage has recently been demonstrated in some angiosperm species (Garcia et al. 2009). The genes are highly conserved even between eukaryotes and prokaryotes, whereas divergence of spacer regions is sufficient to resolve species relationships within most genera (Alvarez and Wendel 2003; Feliner and Rossello 2007). Many hybrids and allopolyploids suffer from numerous rDNA rearrangements (Volkov et al. 2007) including repeat and locus loss (Lim et al. 2007; Weiss-Schneeweiss et al. 2007; Shcherban et al. 2008; Bao et al. 2010; Kotseruba et al. 2010), interlocus recombination and repeat replacement (Wendel et al. 1995; Volkov et al. 2007; Winterfeld et al. 2009). On the other hand, there are several examples of independent nonconcerted evolution of rDNA (O'Kane et al. 1996; Ritz et al. 2005; Kovarik et al. 2008) with a conservation of rDNA loci of progenitors including Brassicaceae (Delseny et al. 1990; Bennett and Smith 1991). Investigations in several synthetic systems demonstrated that changes may occur in early generations after the allopolyploidy event. Yet, their transgeneration dynamics, contribution to stabilization of allopolyploid nucleus, and comparisons with natural populations remain largely unexplored, and we addressed this aspect in the present paper. Epigenetic silencing of parental genes is an important feature of newly established allopolyploids believed to reconcile regulatory incompatibilities between parental subgenomes (Adams and Wendel 2005; Birchler et al. 2005). Epigenetic changes at the rDNA loci following interspecific hybridization have been initially discovered through cytological observations of inheritance of chromosomal secondary constrictions almost 80 years ago (Navashin 1934). Later studies revealed that rRNA silencing depends on both genetic (Flavell 1986; Komarova et al. 2004) and epigenetic factors including DNA and histone modifications (Chen and Pikaard 1997a; Chen et al 1998) and siRNA pathway (Preuss et al. 2008). We hypothesized that active loci with less DNA methylation are more vulnerable to homogenization than the silent loci (Kovarik et al. 2008). Permanently silenced loci may become eventually lost. We therefore investigated copy number and methylation changes in the rDNA units over several generations of synthetic Brassica napus allopolyploid. B. napus genome (AACC, 2n=38) formed recently less than 1,000 years ago by interspecific hybridization of diploid
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progenitors close to modern Brassica rapa (AA, 2n=20) and Brassica oleracea (CC, 2n=18). Both natural populations and resynthesized lines of B. napus display numerous alterations from parental additivity at genomic (Song et al. 1995; Gaeta et al. 2007), transcriptomic (Gaeta et al. 2007), and proteomic (Albertin et al. 2006) levels. Fluorescence in situ hybridization (FISH) was used in karyotype characterization providing valuable information on genome organization and taxonomy in Brassica spp. (Maluszynska and Heslop-Harrison 1993; Snowdon et al. 1997; Kulak et al. 2002; Xiong and Pires 2011). Populations of natural B. napus show genotypic differences in number, distribution, and morphology of rDNA chromosomal loci (Hasterok et al. 2001; Ali et al. 2005; Hasterok et al. 2006). Such variation may have two explanations. The rDNA polymorphisms could stem from ancestral variation given that B. napus is a polyphyletic species (Song and Osborn 1992) and may have multiple origins. Certainly, a variation in loci numbers has been observed in progenitor B. rapa and to a lesser extent between B. oleracea diploids (Hasterok et al. 2006). On the other hand, variability could be induced by interspecific hybridization and polyploidization since changes in locus numbers have been observed in synthetic Nicotiana (Skalická et al. 2003), Arabidopsis (Chen et al. 1998; Pontes et al. 2004), wheat (Shcherban et al. 2008; Baum and Feldman 2011), and Tragopogon (Malinska et al. 2010) allopolyploid forms. To determine genetic and epigenetic dynamics of rDNA loci in Brassica allopolyploids during the first generations following hybridization, we resynthesized B. napus from progenitor species from a homogeneous genetic background using two doubled-haploid lines of the two progenitors, B. oleracea “HDEM” and B. rapa “Z1”. Two S0 synthetic lines were obtained through colchicine doubling (CD) or female unreduced gametes (UG), resulting in four lineages with three selfing generations (S1, S2, S3). We combined structural and functional analyses on the same plants. We carried out rDNA–FISH analysis with the 35S and 5S probes combined with a BoB014O06, BAC clone from the B. oleracea BAC (BoB) library, which hybridizes specifically to all C-genome chromosomes and allows visualization the C-genome for each B. napus plant (Leflon et al. 2006; Nicolas et al. 2009). Homoeologous gene inheritance and their expression were assessed by restriction fragment length polymorphism combined with Southern hybridization and cleaved amplified polymorphism (CAPS). Bisulfite sequencing was applied to determine methylation status of polymerase I promoters. Evidence was obtained for rapid elimination of a subset of rDNA loci following the first meiosis and for unidirectional epigenetic silencing established already in F1 hybrids. Plants of this first selfing generations after hybridization were compared to a natural B. napus.
Author's personal copy Chromosoma (2011) 120:557–571
Materials and methods Plant material Allotetraploid synthetic lines were produced by A-M. Chèvre et al. (unpublished data) by crossing doubled haploid lines, B. oleracea DH (“HDEM”) and B. rapa “Z1” (kindly provided by AAFC, Saskatoon, Canada) as maternal and paternal genome donors, respectively. Reciprocal crosses were unsuccessful. The genealogy of lines is shown in Fig. 1. The single parental F1 hybrid was doubled using colchicine to give a doubled S0 plant. Upon selfing, the progeny gave rise to two sister lines called “CD”. In the second experiment, the same parental F1-producing female unreduced gametes were crossed to the S0 CD as male. The progeny of this cross gave rise to two sister “UG” lines (Fig. 1); we checked in meiosis that all the plants had 2n= 38. For each of the four lines, three selfing generations (S1– S3) were analyzed. The Korean line of B. napus, Yudal, was used as a control. Each plant of the genealogy was maintained by cuttings allowing assessment of the dynamic across the selfing generations (S1–S3). Three cuttings per plant randomized in three blocks were grown under controlled conditions (18°C during 8 h night and 21°C during 16 h day). Samples of leaves, roots, and flower buds just before anthesis were taken and bulked from the three cuttings per plant from 10 to 12 am. Fluorescence in situ hybridization The BoB014O06 BAC clone from B. oleracea BAC library was used as a probe for the C-genome (Alix et al. 2008). This substitute genomic in situ hybridization-like BAC label hybridized specifically to regions on every C-genome chromosome in previous B. napus FISH experiments (Leflon et al. 2006; Nicolas et al. 2007, 2009). The
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BoB014O06 clone was labeled by random priming with biotin-14-dUTP (Invitrogen, Life Technologies). For ribosomal genes, we followed the nomenclature allowing attribution of each chromosome to a linkage group in B. rapa (Kim et al. 2009) and B. oleracea (Howell et al. 2002) (www.brassica.info). The ribosomal probes used in this study were 26S rDNA (Unfried and Gruendler 1990) and pTa794 (Gerlach and Dyer 1980), which contained the 5S rDNA. The 26S rDNA, which was used for detection of 35S rDNA loci, was labeled with digoxigenin-11-dUTP by nick translation and pTa794 with tetramethyl-rhodamine-5dUTP (Roche) using polymerase chain reaction (PCR). The FISH procedure was adapted from Hasterok et al. (2001) with minor modifications (Książczyk et al. 2010). Biotinylated probe was immunodetected by Texas Red avidin DCS (Vector Laboratories) and the signal was amplified with biotinylated anti-avidin D (Vector Laboratories) and digoxigenin-labeled probes were detected with antidigoxigenin antibody conjugated with fluorescein isothiocyanate (Roche). Fluorescence images were captured using a CoolSnap HQ camera (Photometrics, Tucson, Ariz) on an Axioplan 2 microscope (Zeiss, Oberkochen, Germany) and analyzed using MetaVue™ (Universal Imaging Corporation, Downington, PA, USA). Southern blot analysis of 35S rDNA Genomic DNA was extracted either from fresh leaves by a standard CTAB method described by Saghai-Maroof et al. (1984). Southern blotting followed the protocol described by Koukalova et al. (2010) using rDNA probes labeled with [α-32P]dCTP (Izotop, Hungary) in a random-primed reaction (DekaPrime kit, Fermentas, Lithuania). For gene ratio estimation, the probe was a cloned ITS1 subregion obtained by amplification of B. napus DNA with the 18Sfor and 5.8Srev primers (Matyasek et al. 2007). For the DNA methylation analysis, the probe was a 3′ fragment of the 26S gene (Lim et al. 2000). The hybridization signals were visualized by Phosphor imaging (Storm, Molecular Dynamics Sunnyvale, CA, USA) and signals were quantified using ImageQuant software. Expression analysis
Fig. 1 Schematic draws showing genesis of allotetraploids lines
Total RNA was isolated from roots, leaves, and flower buds with aid of RNAeasy kit (Qiagen, Germany) according to the manufacturer’s recommendation. RNA samples were treated with Turbo DNase I (Ambion, Austin, Texas, USA; 0.1 U/μg RNA; 30 min/37°C) to eliminate any contaminating genomic DNA. Quality of RNA preparations was checked with agarose gel electrophoresis. The cDNAs were prepared in a reverse transcription reaction (20 μl) typically containing 1 μg of total RNA, 2 pmols of random primers
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(N9), and 200 units of reverse transcriptase (Invitrogen Superscript II RNase H) following conditions recommended by the supplier (Invitrogen, Paisely, UK). One microliter of cDNA template was used to amplify the ITS1 region using the 18Sfor and 5.8Srev primers (Matyasek et al. 2007). The PCR products were digested with RsaI restriction enzyme allowing discrimination between A- and C-genome transcripts. The fragments were separated on a 2.5% agarose gel.
were grouped into separate FASTA files according to species-specific polymorphisms (ESM 1). These files were statistically evaluated using a CYMATE program (Hetzl et al. 2007).
Results Structure of rDNA in diploid parents and natural B. napus
Bisulfite sequencing Modification of DNA with bisulfite was carried out using the EPITEC kit (Qiagen, Germany) following procedures described by Krizova et al. (2009). The sequencing primers were designed according to the conserved regions not discriminating between A- and C-genome and between methylated and nonmethylated templates. The forward primer (5′-ATGGTTAGAAGAAAAGAAAWTTAT GAAAATTTA-3′) is located at -182 (according to the B. oleracea sequence, Genebank #X56978, Bennett and Smith 1991); reverse primer (5′-TCCCRACCATCRAACCT CAACCCA-3′) was at +111. About 25 clones per sample were sequenced. The A-genome clones were overrepresented by about 50% in hybrids. To roughly maintain an equal number of A- and C-genome sequences, we selected the Cgenome clones by digestion of inserts with SspI. The sequences were aligned (Bioedit, Hall 1999), manually edited, and unambiguous sites were corrected according to the consensus. Sequence heterogeneity caused by mutations (outsides of methylation motifs) was low (
