Plant materials
A set of 40 accessions of M. officinalis have been characterized, of which 27 and 13 were provided from the Federal ex-situ Collection of Agricultural and Horticultural Plants of the Leibniz Institute for Plant Genetics and Crop Plant Research at Gatersleben, Germany (IPK) and the Bavarian State Institute for Agriculture at Freising, Germany (LfL) respectively. LfL collection contained old varieties and breeding material from middle and western Europe, the IPK collection includes mainly landraces and wild types from eastern and middle Europe (Table 1). All IPK accessions were grown from seeds whereas all LfL accessions were propagated by cuttings starting with a single plant. Radish (Raphanus sativus L.) was used as genome size marker in flow cytometry.
Evaluation of ploidy level by flow cytometry
Measuring of relative DNA amount of nuclei occurred by flow cytometry (Facs calibur, Becton Dickinson, BD) with a red fluorescence laser as basis for detection of ploidy level. For each probe, leaf material was chopped with razor blades in 500 μl nuclei extraction buffer (CyStain PI absolute P, Sysmex) and stained with the corresponding staining buffer, containing 5 % polyvinylpyrrolidone 25 (Serva) and 0.6 % propidium iodide (Serva). Immediately after staining, the nuclei suspension was filtered using a 5 ml polystyrene round-button tube with a cell-strainer cap (BD). For reference, radish was measured in separate sample after five samples of balm.
Chromosome preparation
M. officinalis seeds were germinated and the cell division synchronized with 1.25 mM hydroxyurea for 17 h. For vegetative multiplied accessions (LfL), root tips from potted plants were used. In contrast to Pan et al. [16], the recovery time after hydroxyurea treatment was 24 h at 6 °C. Root tips were fixed in ethanol-acetic acid (3:1, 24 h) and stored in 70 % ethanol at −20 °C. After washing with aqua dest. root tips were digested with an enzyme mixture (4 % cellulase, ‘Onozuka R-10’, Serva and 1 % pectlyase Y-23 (Seishin Pharmaceutical)) in 75 mM KCl and 7.5 mM Na-EDTA, (pH 4.0 for 36 min. at 37 °C, [12]). Softened root tips were squashed in 45 % acetic acid. After removal of the coverslip by freezing (−84 °C) the slides were air dried overnight at 24 °C and stored at −20 °C.
Fluorescence in situ hybridization
The 18S-5.8S-25S rDNA loci were detected with a 220 bp-long 25S repeat-specific probe labelled with biotin-16-dUTP (Boehringer Mannheim) during polymerase chain reaction (PCR) amplification of the genomic DNA of Allium ampeloprasum with primers designed according to the sequence published by Yokota et al. [21]. For the localization of 5S rRNA genes, a 117 bp fragment obtained after PCR amplification from the same genomic DNA with specific primers coding for this region [8] was used. The labelling of this amplified probe was performed with digoxigenin-11-dUTP (Boehringer Mannheim). The hybridization mixture contained 80 ng of each DNA probe (5S and 25S rDNA) and 10 μg of salmon-sperm DNA in 20 μl of hybridization buffer (50 % deioinized formamide, 10 % dextran sulphate, 2 x SSC) per slide [19].
The FISH procedure was performed according to Fuchs and Schubert [7] with the following modifications: prior to hybridization, slides were incubated in 50 ng/μl of DNase-free RNase in 2 x SSC for 1 h at 37 °C, washed three times in 2 x SSC for 5 min and treated with 0.5 ng/μl of proteinase K for 10 min at 37 °C, followed by three times washing in 2 x SSC for 15 min. The slides were then postfixed in 4 % paraformaldehyde for 10 min, washed three times in 2 x SSC for 15 min, dehydrated in a graded ethanol series (70, 80 and 96 %) at −20 °C, and air-dried. The hybridization mixture (probe) was denaturated (80 °C, 7 min), incubated on ice (about 5 min), dropped onto slides, covered with coverslips, and sealed with rubber cement. Probes and chromosomes were denaturated together on a heated desk (7 min, 80 °C). The slides were then incubated overnight at 37 °C in a humidity chamber. After hybridization and removing the coverslips, the slides were washed in 2 x SSC at 37 °C three times for 5 min each, followed by three 5 min stringent washes in 0.3 x SSC at 60 °C and then blocked for 30 min at 37 °C with a solution of 4 x SSC, 3 % BSA and 0.1 % Tween 20. The biotinylated probe was detected with 10 ng/μl of streptavidin-Cy3 (Dianova) and amplified with two steps of 10 ng/μl of biotinylated anti-streptavidin (Vector) and 10 ng/μl strepavidin-Cy3. Together with the first amplification step of the biotin labelled probe, the detection of the digoxigenin labelled probe with 9 ng/μl of anti-digoxigenin-fluorescein (Roche) was done and then amplified with 8 ng/μl anti-sheep-fluorescein (Dianova). Chromosomes were counterstained and embedded in 15 μl of DAPI-VECTASHIELD antifade solution (Vector Laboratories). Images were captured for each fluorescence dye separately with a cooled CCD camera system Axiocam (Zeiss) on a microscope Axioimager Z1 (Zeiss) with the following filter combinations: 02 (DAPI), 10 (FITC) and 20 (Cy3). Pseudocoloration and mergence of images were done with software of the Isis program (Metasystems).
