Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism

Arabidopsis Growth Conditions

Arabidopsis (Arabidopsis thaliana) seeds were stratified in water in the dark at 4°C for 2 d before sowing on soil/perlite 3:1 mixture (ED 63, Premium Perlite). Pots were covered with a transparent lid until cotyledons were fully developed and first primary leaves visible. Plants were grown under long-day conditions in controlled-environment rooms (16 h of light, 8 h of darkness, 60-80% humidity, 21°C, 15,550 lux, T5 Tube illumination) or under short day conditions (8 h of light, 16 h of darkness, 60-80% humidity, 21°C, 15,550 lux light intensity).

Arabidopsis lines used in this study were as follows: com1-1 (Uanschou et al., 2007), mre11-4 (Šamanić et al., 2016), hda6-6 (Murfett et al., 2001; Probst et al., 2004; Earley et al., 2006), atxr5-1 atxr6-1 (Jacob et al., 2009), pch2-1 (Lambing et al., 2015), nuc2-2 (Durut et al., 2014), spo11-2-3 (Hartung et al., 2007), lig4-4 (Heacock et al., 2007), and rad51-1 (Li et al., 2004).

Selection of Transgenic Lines

Arabidopsis plants were transformed by the floral dip method (Clough and Bent, 1998). Transgenic plants carrying a BASTA resistance gene were selected from a population by spraying the respective herbicide (BASTA, 1:750 in distilled water, Bayer CropScience) when plants had at least two true leaves. Spraying was repeated every other day until survivors were clearly distinguishable from nonresistant plants. Transgenic lines carrying the Venus-yellow fluorescent protein gene under the At2S3 seed storage promoter were selected under a fluorescence stereomicroscope by collecting seeds positive for yellow fluorescent protein fluorescence.

Generation of an Antibody Directed against Arabidopsis ASY1

The full-length coding sequence of ASY1 was amplified using primers ASY1-XhoI-fwd and ASY1-PacI-rev (primer sequences in the Supplemental Table) and cloned into pFastBac HT A vector for expression in Hi5 insect cells. Both insert and vector were digested with XhoI and PacI restriction enzymes. Transformation and expression of ASY1 was performed at the Vienna BioCenter Facilities (VBCF) protein technologies facility.

The cell pellet was frozen at −80°C and thawed on ice. Subsequent resuspension of the pellet in ice-cold buffer A (1-g pellet per 5 mL of 50 mM phosphate buffer, pH 8, 500 mM NaCl, 2 mM β-mercaptoethanol, 0.05% Nonidet P-40, complete Mini EDTA-free; Roche) was followed by sonication on ice, and an additional round of centrifugation (20,000g, 30', 4°C) yielded a precleared lysate that was incubated with Ni-beads (Profinity IMAC Ni-charged resin; Bio-Rad) for 1 h at 4°C. The Ni-beads were washed three times with buffer A, and the recombinant 6×His-tagged proteins were eluted with 1 M imidazole, pH 8, in buffer A. The purity and concentration of the recombinant proteins in the eluates were analyzed by SDS-PAGE using Coomassie Brilliant Blue R 250 staining and Bradford assay according to standard techniques. After removal of imidazole, the protein solution was concentrated with the help of Vivaspin 20 10,000 MWCO PES centrifugal concentrators (Sartorius Stedim Biotech), and gel filtration was performed using an ÄKTA fast protein liquid chromatography system and a Superdex 200 16/60 column (GE Healthcare Life Sciences). The fractions that contained the His-tagged ASY1 protein were pooled, and ∼0.5 mg of the affinity purified recombinant protein was used for antibody production performed by Eurogentec (Belgium).

Generation and Characterization of Arabidopsis ErDNA Lines

Arabidopsis plants were transformed via the floral dip method with Agrobacterium tumefaciens cells containing the R4 binary vector. Transformed plants were selected on Murashige and Skoog plates (7 g/L bacto agar, 500 mg/L carbenicillin, 12 mg/L glufosinate ammonium) supplemented with 30 µg/mL hygromycin B. Resistant plants were transferred from the plates to soil pots and placed in growth chambers. T3 lines that segregated 3:1 were selected as single insertion lines. To map the location of the ErDNA insertion, a thermal asymmetric interlaced PCR (TAIL-PCR) was performed using primers TAIL A2, TAIL B2, and TAIL C2 together with a random primer, AD7, as shown by Liu et al. (1995). DNA fragments of the expected size were sequenced by Sanger sequencing, and the integration site was subsequently confirmed by PCR (ErDNA2, ectopic rDNA on chromosome 2, six copies integrated at nucleotide position 1,704,488; ErDNA3, ectopic rDNA on chromosome 3, one copy integrated at nucleotide position 10,863,135; ErDNA5, ectopic rDNA on chromosome 5, two copies integrated at nucleotide position 4,999,535).

Expression Analysis of Endogenous rDNA Variants and Ectopically Integrated rDNA Repeats

Expression analysis of rDNA variants (length polymorphism in the 3′-ETS; Pontvianne et al., 2010) was performed by RT-PCR utilizing staged PMCs. Inflorescences from primary and secondary shoots were collected, and each bud was dissected individually. Anthers were modestly squashed to force the release of columns (meiotic cells in syncytia). These were collected in artificial pond water (0.5 mM NaCl, 0.2 mM NaHCO₃, 0.05 mM KCl, 0.4 mM CaCl₂ [Miller and Gow, 1989]). All meiocytes in prophase I were pooled. Anthers that released single entities (dyads and tetrads) were grouped as meiosis I and II. Twenty to 30 anthers per category were collected and frozen immediately in liquid nitrogen. To extract RNA, the SV Total RNA Extraction Kit (Promega) was used following the product specifications. For reverse transcription of the RNA to cDNA, the iSCRIPT cDNA synthesis Kit (Bio-Rad) was used following the product specifications. Finally, to amplify the different rRNA/rDNA variants, a specific primer pair (3allrRNAVAR and 5allrRNAVAR) was used, as described by Pontvianne et al. (2010).

The specific expression of ErDNA was determined via RT-PCR using primers (BstEII-R4Tag-Fw and rDNA-in-rv) directed against a short, integrated stretch of sequence (also including a BstEII site) in the 25S rDNA. The BstEII site served as an ErDNA-specific primer landing platform to exclusively amplify rRNA transcribed from the ectopically integrated rDNA repeat(s). In all cases, expression of the ACTIN-7 (AT5G09810) gene served as reference (actin_ampl3_dn and actin_ampl3_up; see the Supplemental Table for primer sequences).

Determination of Endogenous and Ectopic rDNA Repeat Numbers

DNA was extracted by crushing leaves in UREA buffer (0.3 M NaCl, 30 mM TRIS-Cl, pH 8, 20 mM EDTA, pH 8, 1% [w/v] N-lauroylsarcosine, 7 M urea) and subsequently purified with phenol:chloroform:isoamylalcohol (25:24:1). The qPCR reaction was performed using the KAPA SYBR FAST kit following the product specifications. To quantify 45S rDNA copy numbers, 20–30 ng of genomic DNA was used together with one primer pair (18SRealdn and 18SRealup) as previously described for Arabidopsis (Muchová et al., 2015). The amplification of the rDNA and of the reference gene ACTIN-7 was performed in separate wells and in three technical replicates. The analysis was conducted with four separate biological replicates. The conditions used for the qPCR were 95°C 1 min initial denaturation, 95°C 30 s, 55°C 30 s, 72°C 30 s for 40 cycles with fluorescence detection after every elongation step. The PCR products were not longer than 250 bp and contained a GC content of ∼50%. The experiment was performed on an Eppendorf Realplex 2 Mastercycler. Rosette leaves of different sizes, representing different ages, were collected from bottom to top as follows: t0 = 3.5-cm leaves, t1 = 2.5 cm, t2 = 1.5 cm, t3 = 1 cm for short-day conditions, and t0 = 2.5-cm leaves, t1 = 1.5 cm, t2 = 1 cm for long-day conditions.

For the plant lines containing the ectopic rDNA repeats, those with single insertion sites in the genome were selected according to their segregation patterns. The copy numbers of integrated rDNA repeats at these single insertion sites were determined by qPCR (again taking advantage of the short, integrated stretch of sequence including a BstEII site in the 25S rDNA). To normalize the qPCR results and have an absolute quantification of gene copy number, we used primers (HPT_up_reverse and HYG_down) hybridizing to the hygromycin resistance gene and the previously characterized C2R line (Mittelsten Scheid et al., 2003), which harbors only a single T-DNA at a single integration site. See Supplemental Table for primer sequences.

Preparation of Pollen Mother Cells DAPI Spreads

Inflorescences were harvested into fresh fixative (3:1 96% [v/v] ethanol [Merck] and glacial acetic acid) and kept overnight (O/N) for fixation. Once the fixative decolorized the inflorescences, they were placed in fresh fixative (can be stored for over a month at −20°C), and subsequently, one inflorescence was transferred to a watch glass. The yellow buds were removed to collect only white and transparent buds. The white buds were separated from the inflorescence and grouped according to size. This step is necessary to obtain preparations with separated meiotic stages.

Afterward, the buds were washed three times with citrate buffer (0.455 mL of 0.1 M citric acid, 0.555 mL of 0.1 M trisodium citrate in 10 mL of distilled water) and submerged in an enzyme mix (0.3% w/v cellulase, 0.3% w/v pectolyase in citrate buffer). Each bud has to be submerged for the digestion to work efficiently. The buds were incubated for 90 min in a moisture chamber at 37°C. Digestion was inhibited by adding cold citrate buffer (buds can be kept O/N at 4°C). At this point, the buds were transferred (maximum three to four buds of the same size) to a glass slide. Excess liquid was removed and 15 μL of 60% acetic acid added. The buds were suspended using a metal rod and an additional 10 μL of 60% acetic acid was added to the suspension. The droplet area was labeled using a diamond needle and fixed with fixative 3:1. Slides were dried for at least 2 h. To stage the meiocytes, 15 μL of 2 µg/ml 4’,6 diamidino-2-phenylindol (DAPI) diluted in Vectashield (Vector Laboratories) was added to the slide and sealed with a glass cover slip. Images were taken on a Zeiss Axioplan microscope (Carl Zeiss) equipped with a mono cool-view charge-coupled device camera (Vignard et al., 2007).

Fluorescence in situ Hybridization and Detection of DNA–RNA Hybrids

The DAPI slides selected for FISH were washed in 100% ethanol until the cover slips could be easily removed (5–10 min) and subsequently washed in 4T (4× SCC and 0.05% v/v Tween20) for at least 1 h to remove the mounting medium.

After washing the slides in 2× SCC for 10 min, they were placed in prewarmed 0.01 M HCl with 250 μL of 10 mg/ml pepsin for 90 s at 37°C. The slides were then washed in 2× SCC for 10 min at room temperature. Fifteen microliters of 4% paraformaldehyde was added onto the slides, covered with a strip of autoclave bag, and placed for 10 min in the dark at room temperature. The slides were then washed with deionized water for 1 min and dehydrated by passing through an alcohol series of 70, 90, and 100%, for 2 min each. Slides were left to air dry for 30 min.

Meanwhile, the probe mix was prepared by diluting 1 μL of probe (2–3 µg of DNA) in a total of 20 μL of hybridization mix (10% dextran sulfate Molecular Weight [MW] 50,000, 50% formamide in 2× Saline Sodium-Citrate [SSC]).

In case the rDNA Locked Nucleic Acids (LNA) probe was applied, only 50 pmol (final concentration) was used per slide. The probe mix was denatured at 95°C for 10 min and then placed on ice for 5 min. Afterward, the probe mix was added to the slide, covered with a glass cover slip, sealed, and placed on a hot plate for 4 min in the dark at 75°C. Finally, the slides were placed in a humidity chamber overnight at 37°C. After hybridization, the cover slips were carefully removed and the slides were treated with 50% formamide in 2× SCC for 5 min in the dark at 42°C. The slides were then washed twice with 2× SCC for 5 min in the dark at room temperature.

To detect DNA-RNA hybrids, the Kerafast antibody [S9.6] (Kerafast #ENH001; 1:50 dilution in blocking solution: 1% BSA, 0.01% NaN₃ [w/v] in 1× PBS) was added to the slides at this step and incubated for 30 min at 37°C. The slides were then washed once for 5 min in 2× SCC, and an anti-mouse Alexa 568 antibody (Abcam #ab175473, 1:400 dilution) was added for 30 min at 37°C. At this point, the slides were washed one additional time in 2× SCC.

Finally, 15 μL of DAPI-Vectashield solution was added to the slide and sealed with a cover slip. Images were taken on a Zeiss Axioplan microscope (Carl Zeiss) equipped with a mono cool-view charge-coupled device camera.

Immuno-FISH (Targeted Analysis of Chromatin Events)

Inflorescences with at least two open flowers from the primary shoots of the plant were collected and placed in a Petri dish with wet filter paper.

The buds were dissected with needles under a dissection microscope, and all anthers with transparent lobes were transferred to a droplet of artificial pond water (0.5 mM NaCl, 0.2 mM NaHCO₃, 0.05 mM KCl, 0.4 mM CaCl₂ in deionized water) on a charged glass slide. Afterward, the anthers were squashed between the tips of dissection forceps to release all meiocytes grouped in syncytia (columns).

The slide was transferred to a light microscope, and columns were collected into a 1.5-mL tube on ice with a glass capillary (Chen et al., 2010). Fifteen microliters of digestion solution (1% cytohelicase, 1.5% Suc, and 1% polylvinylpyrollidone) was added to the meiocyte solution and carefully mixed. The tube was incubated for 10 min in the dark at room temperature.

Digestion was stopped by putting the tube on ice. and 8 μL of digested meiocytes was transferred to a clean and charged microscope slide. Twenty microliters of 2% (v/v) Lipsol (Bibby Sterilin, no. 40023) was added to the droplet and mixed by tilting the slide. After 4 min at room temperature, 24 μL of 4% formaldehyde was added to the slide and left to air dry completely (Kurzbauer et al., 2012).

Dried slides were washed in 2× SSC for 5 min, and the primary antibody mix was added after removing excess liquid. The slides were covered with a piece of autoclave bag and incubated in a humidity chamber at 4°C overnight.

The autoclave bag cover was removed, the slides were washed in 2× SSC for 5 min, and the appropriate secondary antibody mix was added. The slides were again covered with an autoclave bag and incubated in a humidity chamber for 1 h at 37°C.

The slides were washed for 5 min in 2× SCC, and 15 μL of 4% formaldehyde fixing solution was added to the slide, covered with a strip of autoclave bag, and incubated for 10 min in the dark.

Afterward, slides were rinsed by dipping into deionized water and dehydrated by passing through an alcohol series of 70, 90, 100% ethanol, for 2 min each. Finally, the slides were left to dry completely for 30 min in the dark.

To prepare the probe mix, 14 μL of hybridization solution was added to 2–3 μL of Bacterial Artificial Chromosome (BAC) probes (2–3 µg) or 1 μL of 1 µmol LNA probe and filled up to a final volume of 20 μL with deionized water.

The probe mix was denatured for 10 min at 95°C and then placed on ice for 5 min. After cooling, the probe mix was added to the slide and covered with a glass cover slip. The borders of the cover slip were sealed with rubber cement and placed on a hot plate for 4 min in the dark at 75–80°C (75°C for repetitive DNA and 80°C for multiple BACs).

The slides were incubated in a humidity chamber O/N at 37°C. If an LNA probe was used, 3–4 h were sufficient for a successful hybridization.

Thereafter, the slides were washed in 50% formamide–2× SCC for 5 min in the dark at 42°C and twice in 2× SCC for 5 min.

Finally, 15 μL of DAPI-Vectashield was added to the slide and covered with a glass cover slip. The borders of the glass slide were sealed with transparent nail polish. Slides were imaged with a conventional fluorescence microscope (Zeiss Axioplan). Z-stacks with 100-nm intervals were acquired, deconvolved using AutoQuant software (Media Cybernetics), and are presented as projections done with the HeliconFocus software (HeliconSoft). Super-resolution images were acquired using the STEDYCON system (Abberior).

All BACs were labeled using the Nick Translation mix from Roche following the manufacturer’s instructions. Each BAC was labeled individually and concentrated in a tube. In this study, fluorescently labeled nucleotides Chromatide Alexa Fluor 488-5-dUTP (Thermo Fisher Scientific), Chromatide Texas Red-12-dUTP (Thermo Fisher Scientific), and Cy5-dUTP (GE Healthcare) were used.

The dilutions used for the different primary and secondary antibodies are as follows: anti-H3K27me1 1:200, anti-H4(Ac) 1:50, anti-ASY1 1:10,000, anti-RAD51 1:300 (Kurzbauer et al., 2012), anti-ZYP1 1:500 (Higgins et al., 2005), anti-SCC3 1:500 (Chelysheva et al., 2005), anti-REC8 1:250 (Cai et al., 2003), anti-MRE11 1:200 (Lohmiller et al., 2008), anti-guinea pig Alexa 488 1:400 (Abcam #ab150185), anti-rabbit Alexa 568 1:400 (Abcam #ab175471), anti-rat Alexa 568 1:300 (Abcam #ab175476), anti-mouse Alexa 568 1:400 (Abcam #ab175473), anti-guinea pig STAR 580 (for STEDYCON only) 1:500 (Abberior #201120057), and anti-rabbit STAR RED (for STEDYCON only) 1:250 (Abberior #200120119).

Whole Mount Immuno-FISH

This method is an adapted version of the “whole-mount FISH in a tube” from (Bey et al., 2018).

Inflorescences were collected from the primary shoots of an Arabidopsis plant, and all open flowers and pollen-containing buds were removed with forceps and a needle under a dissection microscope.

All remaining buds were opened with the help of two needles to increase accessibility of anthers to the enzyme solution and the FISH probe. The inflorescences were placed in 500 μL of fixation solution (1% formaldehyde, 10% DMSO, 1× PBS, 60 mM EGTA), and all buds were submerged.

The solution with the buds was placed under vacuum for 10 min at room temperature and incubated for a further 30 min at room temperature.

After removing the fixation solution, the samples were incubated 2 × 10 min in 500 μL of methanol.

Methanol was removed, and samples were incubated for 2 × 5 min in 500 μL of ethanol.

Finally, after the ethanol was removed, the samples were incubated in 500 μL of xylene/ethanol (1:1) for 15 min. The samples were transferred to a new tube with 500 μL of xylene and incubated for 30 min at 50°C. Samples were washed twice in 500 μL of ethanol and 500 μL of methanol and washed three times in 500 μL of PBT (1× PBS with 0.01% v/v Tween 20).

The samples were digested by incubating the buds in the enzyme solution (0.6% cytohelicase, 0.6% pectolyase, 0.6% cellulase in citrate buffer, pH 4.5) for 1 h at 37°C.

The samples were gently washed twice in 500 μL of 2× SSC and then incubated in 500 μL of 0.1 mg/ml RNaseA in 2× SSC for 1 h at 37°C. Afterward, the samples were washed twice in 500 μL of 1× PBT and fixed in 1% formaldehyde (in 1× PBT) for 30 min.

Samples were then washed twice in 500 μL of 1× PBT and once in 500 μL of 2× SSC, followed by incubation in 500 μL of a 1:1 mix of HB50 (50% formamide, 2× SSC, 50 mM NaH₂PO₄) and 2× SSC for 30 min, incubated in 500 μL of HB50 for 30 min, and finally incubated in 30 μL of hybridization solution (labeled DNA probe 0.5–1.5 µg for DNA repeats and 2–5 µg for unique sequences, 50% formamide in 2× SSC) in the dark for 1 h.

To denature the probe and the target sequence, the tube was placed in a heating block for 4 min at 85°C and, afterward, on ice for 3 min. Finally, to hybridize the labeled probe to the sample, the tube was left overnight in the dark at 37°C.

The samples were washed in 500 μL of HB50, incubated in 500 μL of fresh HB50 at 42°C for 1 h, and washed in 500 μL of PBT for two times, 10 min each.

To successfully immune label proteins in a whole-mount preparation, once the samples were washed in PBT, they were incubated in 50 μL of primary antibody mix under vacuum for 30 min and then transferred for 3 h to 37°C or overnight at 16°C. The primary antibody mix was 10 times more concentrated than what is usually used for regular spreads. Afterward, the samples were washed four times in 1× PBT and incubated with 50 μL of the secondary antibody solution for 30 min under vacuum and placed for 3 h at 37°C.

Finally, the sample was washed three times for 15 min in 1× PBT, incubated in 100 μL of 1× PBS with 8 μl of DAPI 5 μg/ml for 30 min, and placed on a glass slide with 20 μL of DAPI-Vectashield. Imaging of whole-mount samples was performed with a Zeiss LSM710 equipped with an AiryScan Unit with 160-nm resolution in x, y, and z. The images were deconvolved and 3D rendered with the Huygens Software (SVI software).

Scoring Recombination Rates

Recombination analysis was performed according to the protocol published by Berchowitz and Copenhaver (2008). In brief, individual flowers were collected from the main shoot and tapped on a glass slide with pollen sorting buffer (10 mM CaCl₂, 1 mM KCl, 2 mM MES and 5% [w/v] Suc, pH 6.5) (Yelina et al., 2013) for 2–3 min until all pollen was released. Tetrads were scored with an inverted microscope equipped with three filter sets for green, red, and cyan fluorescent proteins. All plant lines used for tetrad analysis were in the qrt1-2 mutant background.

We flanked the ectopic 45S rDNA integration (ErDNA2) at nucleotide position 1,704,488 on chromosome 2 with FTL 1431 (at nucleotide position 1,521,041) and FTL 2269 (at nucleotide position 8,276,753), ErDNA3 at nucleotide position 10,863,135 on chromosome 3 with FTL 1019 (at nucleotide position 1,517,290) and FTL 1046 (at nucleotide position 9,458,743) and ErDNA5 at nucleotide position 4,999,535 on chromosome 5 with FTL 1143 at nucleotide position 3,760,756 and FTL 2450 at nucleotide position 5,497,513. For all analyses, lines homozygous for the ectopic rDNA insertion sites have been used.

CRISPR/Cas9-Mediated ErDNA Deletion

To specifically delete the rDNA portion of the R4 transgene (Wanzenböck et al., 1997) but retain the resistance marker and the left border and the right border of the vector after integration in the plant genome, a CRISPR/Cas9 vector was constructed as described before (Richter et al., 2018). Two guide RNAs (gRNAs) were designed that bind between the left border of the R4 binary vector and the start of the rDNA sequence and between the 3′ETS and the promoter of the hygromycin-resistant gene, respectively. All gRNAs were designed to contain at least 50% GCs and to be next to Protospacer Adjacent Motif (PAM) sequence. The plants transformed with the Cas9 construct were selected with BASTA and the survivors evaluated for Cas9 activity on both target sites. This was achieved by amplifying, by PCR, the gRNA target regions within the T-DNA, followed by subsequent Sanger sequencing.

Only T1 plants that showed Cas9 activities on both target sites were propagated to the T2 generation and genotyped for loss of the ectopic rDNA unit within the transgene. It was necessary to go through at least two plant generations to select for a homozygous deletion and for loss of the Cas9 transgene.

Definition and Quantification of RAD51 Foci

RAD51 foci were counted manually on deconvolved, slice-aligned, and 16-bit projected images. Only RAD51 foci colocalizing with the chromatin (DAPI) were accepted. Only RAD51 foci overlapping with 50% or more with the genomic region specifically labeled with a FISH probe were scored as a colocalizing event.

Quantification of Fluorescence Intensity

Fluorescence intensity was calculated with Fiji using the profile Plot analysis tool. Fluorescence intensity for each channel was normalized to the highest fluorescent value.

Quantification of rDNA Fragmentation via FISH

rDNA fragments were counted manually on 16-bit images of meiotic cells from metaphase II to the tetrad stage. Only rDNA signals (FISH) that overlapped with a chromatin signal (DAPI) were accepted.

Nucleolus Association of the ErDNA

Whole-mount immuno-FISH images were 3D reconstructed to locate the nucleolus as an empty pocket within the nucleus. The specific genomic regions (FISH signals) were accepted as “nucleolus associated” in case they were located at the borders or within the nucleolus itself. All experiments were performed by also detecting the endogenous 45S rDNA as a reference for the nucleolus location.

Statistical Analysis

All statistical analyses, t Tests, Mann-Whitney test, and binary logistic regression tests (as indicated in the figure legends) were performed using GraphPad Prism software (GraphPad Software) and http://statpages.org/logistic.html.

Unpaired, two-tailed Mann-Whitney tests were performed, since D’Agostino Pearson omnibus K2 normality testing revealed that most data were not sampled from a Gaussian population, and nonparametric tests were therefore required. Error bars indicate standard deviations.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers COM1 AT3G52115, MRE11 AT5G54260, HDA6 AT5G63110, ATXR5 ATXR6 AT5G09790–AT5G24330, PCH2 At4G24710, NUC2 AT3G18610, SPO11-2 AT1G63990, LIG4 AT5G57160, RAD51 AT5G20850.

Supplemental Data

• Supplemental Figure 1. The NORs are highly dynamic regions that are both transcribed during meiosis.

• Supplemental Figure 2. The rDNA acquires distinct chromatin characteristics during meiosis.

• Supplemental Figure 3. The rDNA acquires a specific chromatin environment during meiosis.

• Supplemental Figure 4. The nucleolus shields rDNA from meiotic DSB formation and deleterious HR.

• Supplemental Figure 5. The rDNA is repaired by NHEJ.

• Supplemental Figure 6. Ectopically integrated rDNA units associate with the nucleolus, promote homolog pairing and suppress meiotic recombination.

• Supplemental Table. Primers and BACs used in this study.

• Supplemental Movie 1. In G2 the rDNA is not localized within the nucleolus.

• Supplemental Movie 2. During leptotene the rDNA is localized within the nucleolus.

• Supplemental Movie 3. ASY1 does not localize within the nucleolus and to the rDNA during leptotene.

• Supplemental Movie 4. ASY1 does not localize within the nucleolus and to the rDNA during zygotene.

• Supplemental Movie 5. ASY1 localizes to the NORs of chromosome 2 and chromosome 4 during pachytene.

• Supplemental Movie 6. Nucleolus association of an ectopic rDNA unit (ErDNA).