Arabidopsis thaliana accession Columbia (Col-0) seeds were surface sterilized and grown in vitro at 20°C, 75% humidity, 60 μmol m^-2 s^-1 and under a 10 hours light/14 hours dark period as described previously 17.

We used the following DNA clones: 180 bp pAL1 15, 5S rDNA pCT4.2 16, 45S rDNA 17, Bacterial Artificial Chromosomes (BAC) T6P5 (GenBank Accession AC005970) and T1J1 (GenBank Accession AF128393), located on small arm of chromosome 2 and 4, respectively. T6P5 and T1J1 belong to the tiling paths of BAC clones selected for chromosome painting: they contain <5% of mobile elements and are specific to chromosome 2 and 4 18. DNA was extracted using Nucleobond extraction kit (Clontech) according to the manufacturer's instructions.

Triton X-100.

Dextran sulfate 20%.

RNAse (Sigma AR-6513, stock solution 10 mg/ml).

Klenow fragment (40 U/μl) (Invitrogen Y01396).

BioPrime^® DNA Labelling System (Invitrogen 18094-011: 2.5× random primers solution 125 mM Tris-HCl (pH 6.8), 12.5 mM MgCl₂, 25 mM β-mercaptoethanol, 750 μg/ml oligodeoxyribonucleotide primers (random octamers) (Invitrogen Y01393).

Fluorochromes: ChromaTide^® Alexa Fluor^® 488-5-dUTP (Invitrogen C11397), Cy3-dUTP (Amersham PA53022), Cy5-dUTP (Amersham PA55022). Prepare a 10× fluorophore mixture (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM fluorochrome).

DAPI (4'6-Diamidino-2-phenylindole) at 1 μg/ml final concentration into VECTASHIELD^® antifade mounting medium (Vector Laboratories).

Cryomount medium (Histolab 00890).

Nuclei extraction buffer (10 mM Tris HCl pH 7, 4 mM spermidine, 1 mM spermine, 5 mM MgCl₂, 0.1% triton X-100).

1× Phosphate-Buffered Saline (PBS) pH 7.3 (3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄, 135 mM NaCl, 1.3 mM KCl).

20× SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0).

Stop buffer 0.5 M Na₂EDTA pH 8.0 (Invitrogen 50690).

HB50 (50% deionized formamide, 2× SSC, 50 mM phosphate, pH 7.0).

Vacuum pump.

Centrifuge Sigma 3K18 rotor 11133.

Nanodrop, Labtech spectrophotometer ND-1000.

Hot plate (75–80°C) and heating block (100°C).

Incubators (37°C and 55°C) and a moist chamber.

Coplin jars.

Silanized slides (Dako S3003) or SuperFrost^® Plus glass slides (Menzel-Gläser J1800AMNZ).

Coverslips (22 × 22 and 24 × 50 mm).

Dako pen (Dako S2002).

Cryostat (Leica CM-3050S).

Fix young seedlings (18 days-post-germination) in 4% paraformaldehyde (PFA) in 1× PBS buffer at room temperature (RT) for 30 minutes under a vacuum. Replace the fixative solution with fresh fixative solution and fix for an additional 30 minutes under a vacuum. Rinse seedlings in 1× PBS twice for 5 minutes each and store in 1× PBS at 4°C until use (fixed seedlings can be stored for up to a month).

The extraction protocol is based on the existing protocol reported by 19. All centrifugation steps were carried out at 500 g (1700 rpm) for 3 min, at room temperature.

Place up to 8 seedlings in a 1.5 ml Eppendorf tube containing 500 μl of nuclei extraction buffer. Prior to extraction, add β-mercaptoethanol to a final concentration of 5 mM. Extract nuclei gently with a plastic pestle. Filter the nuclei suspension through a 50 μm nylon mesh. Discard the supernatant after centrifugation and wash the pellet with 1× PBS. Treat the pellet with 300 μl of 1× PBS containing 0.5% triton X-100 for 5 minutes. Centrifuge, discard the supernatant and wash the pellet with 1× PBS for 3 minutes.

Resuspend the pellet in 30 μl of 1× PBS. Add 4 μl of this suspension to a slide and dry the slide at 4°C for 20 minutes before mounting with DAPI or carrying on with the FISH procedure. Draw a circle around the drop of nuclei on the slide using a DAKO pen to avoid deforming the specimen once mounted with a coverslip.

To prepare tissue for cryostat sectioning, cut leaves into small pieces (about 5 mm²) and incubate the leaf sections in 15% sucrose overnight at 4°C. Place a piece of leaf on a slide, remove the excess of sucrose and add to the leaf a drop of mounting medium. Transfer the slide to a cryostat chamber maintained at -25°C during sectioning and leave the slide within the cryostat chamber until the mounting medium and leaf sample are frozen. Remove the frozen sample from the slide with forceps and transfer it to a mounting stub in an upright position for transversal sectioning. Add few μl of mounting medium and correct the orientation of the sample being careful to maintain it in an upright position until the mounting medium freezes.

To section the tissue, transfer the sample to the holder and adjust the orientation of the sample, the microtome knife and the anti-roll plate. For FISH studies, we used 10, 16 and 20 μm sections. After sectioning, carefully place the leaf sections on a SuperFrost or silanized slide that has been pre-cooled to -25°C. The sample can be stored on the slide at -20°C until use. Let the slide thaw at room temperature before washing in 1× PBS for 5 minutes to remove the mounting medium.

Incubate leaf sections with 1× PBS containing 0.5% triton X-100 for 10 minutes or incubate isolated nuclei with 1× PBS containing 0.5% triton for 3 minutes at RT. Wash the slide with 1× PBS for 5 minutes to remove the detergent and proceed to the HCl treatment. Treat leaf sections with sterile water containing 0.1 N HCl for 20 minutes at RT. Rinse the slide with sterile water for 5 minutes and proceed directly to the RNase treatment. For the RNase treatment, equilibrate the slide in 2× SSC for 5 minutes at RT then incubate the slide in 2× SSC containing 400 μg/μl RNase for 15 minutes in a moist chamber at 37°C. Rinse the slide in 2× SSC for 2 minutes at RT. Apply the HB50 prehybridization mixture (~100 μl) to the slide, place a coverslip over the sample and incubate it in a moist chamber at 37°C for 1 to 2 hours.

On ice, add 20 μl of 2.5× random primers solution to 20 μl DNA (100 ng). Denature this mix at 100°C for 10 minutes and immediately cool it on ice. Add 5 μl of fluorochrome mixture and distilled water to a total volume of 49 μl. After a brief vortexing, add 1 μl of klenow enzyme. Mix gently by pipetting up and down and centrifuge the reaction for 30 seconds. Incubate the reaction at 37°C for 1 hour to yield 100 to 200 base pairs (bp) fragments. Load 5 μl of the reaction on a 1% agarose gel to verify the length of the DNA fragments. If the DNA length is not between 100 and 200 bp, incubate for an additional hour or up to 24 hours. Stop the reaction, by adding 5 μl of stop buffer and store the probe at -20°C until use.

Prepare the probe mix as follows: 10 μl of 20% dextran sulfate, 1 to 9 μl HB50 (50% deionized formamide in 2× SSC and 50 mM sodium phosphate, pH 7.0), 1 to 9 μl of probe and adjust to a final volume of 20 μl. Use 100 to 400 ng of DNA for BAC clones and 20 to 100 ng of DNA for repeated DNA. Denature the probe at 100°C for 5 minutes and transfer immediately to ice.

Denature the sample at 75°C for 5 minutes, drain from the denatured slide any excess fluid, and add 20 μl of denatured probe to the sample. Cover the sample with a coverslip and hybridize it in a moist chamber at 37°C for 2 to 3 hours or overnight for convenience.

Wash sample as follows: 2 washes in 2× SSC for 15 minutes each at 55°C; 1 wash in 1× SSC for 10 minutes at 55°C and 1 wash in sterile water for 2 minutes at RT. Mount the slide in 3 μl of Vectashield containing 1 μg/ml DAPI. Seal the coverslip with colorless nail polish and store at 4°C (sealed samples can be stored up to a month at 4°C).

All images were taken with a Leica DMIRE2 SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). A UV diode (405 nm) was used for DAPI visualization and three lasers at 488 nm, 543 nm and 633 nm were used for the excitation of Alexa Fluor^® 488-5-dUTP, Cy3-dUTP and Cy5-dUTP, respectively. Images were taken through a 63× water immersion objective (NA 1.2, WD 0.2 mm). Z series images were collected at 0.120 μm intervals. Images were scanned with a zoom of 9.29, a frame average of 2 and a line average of 2.

Confocal image stacks were automatically converted to a format enabling their processing by programs developed using the Free-D software libraries 20. To separate the nucleus from the background, a preliminary intensity threshold was computed using the isodata algorithm 21. Since this algorithm is sensitive to the relative size of the nucleus within the image, the threshold was generally too high because of the larger background size. This bias was corrected by setting the actual threshold to m-2s, where m and s are the intensity average and standard-deviation, respectively, computed over the nucleus region defined by the preliminary threshold. Holes due mainly to the nucleolus, boundary irregularities due to noise, and bumps due to blur from chromocenters 22 were smoothed out using hole filling, opening, and closing binary morphological operators 23. Finally, a surface model of the nuclear envelope was generated by applying the marching cubes algorithm 24 to the nucleus binary mask.

Nucleus size was quantified from the equivalent spherical diameter, i.e., the diameter of the sphere with the same volume as that enclosed by the nuclear surface. Nucleus global shape was quantified using the compactness parameter: Compactness = 36 π volume²/surface area³.

This parameter, which is comprised between 0 and 1, characterizes shape regularity and takes its maximum value for a sphere. To quantify an eventual flattening of the nucleus, a flatness parameter was derived from the lengths of the principal axes of the nuclear surface: Flatness = length of intermediate axis/length of shortest axis. Flatness was only computed over the nuclei for which the shortest axis corresponded to the Z (optical axis) direction.

Morphological parameters measured on isolated nuclei before or after FISH were compared to measures made on nuclei from leaf sections with the Mann-Whitney test (alpha = 5%) using the R statistical software 25.