Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae

Botanical Journal of the Linnean Society, 2002, 139, 99–108. With 16 figures

INTRODUCTION

Allium commutatum Guss. belongs to the sub-genus Allium, section Allium, the largest and the most widely distributed group centred on the Mediterranean, Oriental and Caucasian floristic re-gions (Hanelt, 1996). Hanelt et al. (1992) describedsubgenus Allium as an actively evolving group inwhich the determination of species is often difficult,

particularly those from sections Codonoprasum andAllium. Gussone (1854) originally described A. com-mutatum Guss. as an endemic species of the Island ofIschia. It has also been described under the names A. ampeloprasum var. lussinense and A. bimetrale inCroatia, Greece and Italy (Haracic, 1894; Bothmer,1970; Capineri, 1971). Bothmer (1974) and laterStearn (1989) used these two names for the first timeas synonyms for A. commutatum. Allium commuta-tum is a diploid species (Garbari & Cela Renzoni,1975; Johnson, 1982) with the exception of some

© 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 139, 99–108 99

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss.(Alliaceae)

V. BESENDORFER1*, M. SAMARDZIJA1, V. ZOLDOS1, M. E. SOLIC2; and D. PAPES1

1Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia2Institute ‘Mountain and the Sea’, HR-21300 Makarska, Croatia

Received June 2001: accepted for publication February 2002

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region(NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic popula-tions of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied,even those of distant islands. Diploid karyotypes (2n = 16) always possessed two NOR-bearing chromosome pairswith pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluo-rochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands wasnoticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was corre-lated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as numberand size of silver stained NORs, existed between cells and between individuals of the same population. The differ-ent size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich consti-tutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed betweenhomologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society,2002, 139, 99–108.

ADDITIONAL KEYWORDS: 18S-5.8S-26S rDNA – 5S rDNA – fluorochrome banding – fluorescence in situhybridization – silver staining.

*Corresponding author. E-mail: [email protected]

triploid and tetraploid Greek populations (Bothmer,1970; Tzanoudakis, 1992). So far, simple karyomor-phological investigations have shown a high level ofkaryotype similarity for species in sect. Allium, espe-cially those belonging to A. ampeloprasum complex(Maggini & Garbari, 1977; Pejcinovic, 1997). The basic chromosome number of this section is x = 8, with the exception of A. heldreichii with x = 7(Mathew, 1996). Karyotypes are characterized by chromosomes with pericentric and median sec-ondary constriction (SCs) positions, described by VedBrat (1965) as ‘sativum’ and ‘scorodoprasum’ types,respectively.

Heterochromatin and other chromosomal regionswith different structural and chemical properties (AT- and GC-rich regions, nucleolar organizing regions(NORs), etc.) can be differentiated by use of modernchromosome banding techniques. Banding patternsmay be valuable in studies of species relationships as heterochromatin organization can vary highlybetween closely related species. Cytochemical char-acterization of constitutive heterochromatin by fluorochrome staining can furthermore help in karyological characterization of species populations,varieties and cultivars (Moscone et al., 1995).

NORs are the chromosomal sites of tandemlyrepeated DNA sequences coding for 18S-5.8S-26S(18S-26S) rRNA. Cytologically, these regions areusually located in SCs of metaphase chromosomes,and in telophase–interphase they are responsible forthe formation of the nucleolus. NORs are usuallyflanked by regions of constitutive heterochromatin ofvarious sizes that can be detected and characterizedby differential banding techniques. NORs can bedetected by silver staining (Goodpasture & Bloom,1975). This technique reveals NORs that are func-tionally active during the preceding interphase, whilethe size of silver deposit is correlated with the degreeof the transcriptional activity or, even more, with the number of rDNA repeats (Cermeño et al., 1984;Mellink, Bosma & De Haan 1994; Zurita et al., 1997).However, the only certain method for identifying allsites of 18S-26S rDNA, whether active or inactive, isin situ hybridization to ribosomal DNA sequences withthe 18S rDNA probe. Another ribosomal gene family,5S rDNA, is usually separated from 18S-26S rRNAgenes on non-NOR-bearing chromosomes.

We have recently reported the karyotype and hete-rochromatin distribution in A. commutatum using C-banding and fluorescent staining (Besendorfer et al.,1997). The haploid karyotype consists of five meta-centric chromosomes (I-V), two submetacentrics (VIand VII) and one subtelocentric (VIII). Chromosomepair VII has SC proximal to the centromere of theshort arm, while the SC in the short arm of chromo-some pair VIII is median in position. By application of

silver staining we confirmed that NORs are located on chromosomes with SCs. The present study wasdesigned to reveal the number of chromosomal loci ofboth ribosomal gene families, 18S-26S and 5S rRNAgenes, by means of fluorescent in situ hybridization(FISH). The hybridization pattern is also comparedwith the distribution of heterochromatin in metaphasechromosomes of several natural populations of A. com-mutatum. Cell-to-cell and specimen-to-specimen dif-ferences of NOR-activity are analysed and discussedwith respect to FISH results.

MATERIAL AND METHODS

PLANT MATERIAL

The analysis was carried out on six south-easternAdriatic populations of A. commutatum (Fig. 1). Thebulbs of four populations were collected on three southAdriatic islands, Palagruza, Vis and Bisevo, during thesummers of 1996 and 1997. The plants from two main-land populations, Kastela and Makarska, were col-lected during the spring of 1997 and cultivated in theBotanical Garden of the Faculty of Science, Universityof Zagreb.

CHROMOSOME PREPARATIONS AND

GIEMSA C-BANDING

The roots were pretreated with 0.05% colchicine(Sigma) at room temperature for 3–4h, and fixed in3 :1 ethanol-acetic acid. For Giemsa C-banding, twomethods of Gill et al. (1991) with slight modification(Besendorfer et al., 1997) were applied. Chromosomepreparations for fluorochrome banding, silver stainingand FISH were carried out by enzymatic macerationin a mixture of 20% pectinase (Sigma) and 2% cellu-lase (Calbiochem) in 0.1M citrate buffer at 37°C for40min. After maceration the root-tips were squashedin 45% acetic acid. Preparations in which the chromo-somes were outside the cell wall were selected forFISH treatment. The coverslip was removed from theslide by the dry-ice method (Sharma & Sharma, 1972).

FLUOROCHROME BANDING AND SILVER STAINING

For fluorescent banding Chromomycin A3 (CMA)(Sigma), 4,6-diamidino-2-phenylindole (DAPI) (Sigma)and Hoechst 33258 (Sigma) were used. The modifiedstaining procedure of Schweizer & Ambros (1994) wasapplied. Preparations were stained with 0.1mgmL-1

CMA for 60min. Slides were stored for 2–3 days at37°C prior to examination under a Zeiss Opton Axio-plan epifluorescent microscope. The same slides werede-stained from CMA and re-stained with 0.2mgmL-1

DAPI for 12min. Some preparations were stained onlywith 0.1mgmL-1 Hoechst.

100 V. BESENDORFER ET AL.

© 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 139, 99–108

rRNA GENES IN ALLIUM COMMUTATUM 101


Figure 1. Locations of the collected south-eastern Adriatic populations of Allium commutatum.

Silver staining of NORs and nucleoli was performedas described by Hizume, Sato & Tanaka (1980). Silver-impregnated slides destined to be stained further with CMA were de-impregnated according to Berg &Greilhuber (1992). Silver stained nucleolar organizingregions (Ag-NORs) and nucleoli were studied on thesame slides. Approximately 500 interphase cells wereanalysed for each population except for that fromPalagruza, where up to 4000 were scored.

FLUORESCENCE IN SITU HYBRIDIZATION

Two ribosomal gene families were used as probes for in situ hybridization: the probe 3–3750–25S, con-taining a 4kb EcoRI fragment with almost entire 18SrDNA, isolated from Arabidopsis thaliana and clonedin pSK plasmid (provided by Prof. D. Schweizer, Institute of Botany, Vienna), and the probe pTa794 containing a complete 410bp BamHI fragment of 5S rDNA that was isolated from wheat and cloned in pBR322 (Gerlach & Dyer, 1980). The probe3–3750–25S was labelled with digoxigenin-11-dUTP(Boehringer-Mannheim) and the pTa794 with Fluoro-

Red-dUTP (Amersham) by the polymerase chain reac-tion (PCR). Denaturation of chromosomal DNA andthe DNA of the probes, DNA:DNA hybridization,posthybridization washes, and the single detectionstep with antidigoxigenin coupled with FITC werecarried out as described by Heslop-Harrison et al.,1991) with the slight modification described byCerbah, Coulaud & Siljak-Yakovlev (1998). For bothsimultaneous and one-step FISH, the DNA probeswere mixed to a final concentration of 40–60ngmL-1

in a solution of 50% (v/v) formamide, 10% (w/v)dextran sulphate, 0.1% (w/v) sodium-dodecyl sulphate,250mgmL-1 salmon sperm and 2xSSC. Slides werecounter-stained with DAPI (2mgmL-1) and mounted inantifade solution (AF1 citifluor). Preparations wereexamined under a Zeiss Axioplan microscope withappropriate filter sets.

Hybridization signals were analysed from the fluo-rescent images directly frozen in the image framememories of an image analyser by means of a highlysensitive CCD camera (Sony). The chromosomes’superimposed images were three-coloured contrastmanipulated but not otherwise processed.

RESULTS

DISTRIBUTION AND CHEMICAL PROPERTIES

OF HETEROCHROMATIN

The karyotypes of all of the populations studied had2n = 2x = 16 chromosomes. After Giemsa C-banding,marker C-bands appeared at the SCs of the two NOR-bearing chromosome pairs VII and VIII, and in anintercalary position in chromosome pair VI (Fig. 2).The dimorphism, visible as different staining inten-sity, was more prominent for the homologues of theNOR-bearing pair VII and of the pair VI. The stainingintensity of C-bands differed from very prominent C-bands at the SCs to weak centromeric and telomericones (Fig. 2). The most prominent telomeric C-bandwas that on the satellites of pair VIII.

Chromosomes were first stained with CMA and sub-sequently re-stained with DAPI. CMA-positive NOR-associated heterochromatin was detected at the SC ofchromosomes VII and VIII that corresponded to thelocation of the C-bands (Figs 2, 3). These CMA bandswere slightly different in size between homologues. Ina few metaphases weak DAPI- and H-positive bands,corresponding to intercalary C-bands, were noticed onchromosomes of the pair VI. DAPI subsequent to FISHproduced DAPI signals in the centromeric regions ofall chromosomes (Figs 11, 15), in the telomeric regionon the satellites of pair VIII (Fig. 15) and in SCs ofpairs VII and VIII (Figs 14, 15).

ACTIVITY OF NORS

Silver staining revealed metaphase plates with dif-ferent number of Ag-NORs in the same individualand/or populations (Figs 4–9). Cells with the maximalnumber of four Ag-NORs located in SCs of chromo-some pairs VII and VIII (Figs 4–7), as well as cellswith one, two or three Ag-NORs, were recorded(Figs 8, 9). Table 1 shows that two, three and four Ag-NORs appeared more frequently (in 26.8%, 31.1% and31.6% of the cells scored, respectively) than only oneAg-NOR (in 10.5% of the cells scored). The exceptionwas the Palagruza population, in which cells with twoand three Ag-NORs were more frequent, and the

Makarska population, in which there was no dominantnumber of active NORs.

Most of the metaphases with two Ag-NORs werelocated on one of the homologues of each NOR-bearingpair. Forty out of 71 metaphases with three Ag-NORshad active NORs on both homologues of the pair VIII.We were also interested to see whether there is a pre-dominance of activity of heterologous NORs and whichof the two NORs is more frequently active. Table 1shows that NORs of pair VIII were active in more cells(53.39% of total Ag-NORs) then those of pair VII(46.61% of total Ag-NORs). In the Komiza, Bisevo,Kastela and Makarska populations, NORs of pair VIIIwere active in more cells, while in the Palagruza population NORs of the pair VII were more frequentlyactive and in the Vis population equal activity of bothNORs was recorded.

Variation of Ag-NOR size within and betweenmetaphases of the same individual was observed.Dimorphism existed between nonhomologous as wellas homologous NORs (Figs 4, 6, 7, 9). In the most casesthe size of Ag-NORs corresponded to the extension ofSCs. In any population or individual studied it waspossible to find cells with four nucleoli. However, moreoften than not this maximum number of nucleoli wasfound in the cells (Fig. 10). Table 2 shows that inter-phases with two and three nucleoli appeared more frequently than those with one or four nucleoli.

NUMBER AND CHROMOSOMAL LOCATION OF

18S-26S AND 5S rRNA GENES

Simultaneous as well as one-step in situ hybridizationusing appropriate rDNA probes was applied to deter-mine the position and number of the 18S-26S rDNAand 5S rDNA loci in A. commutatum. Four FITC greenhybridization signals, corresponding to two loci,appeared at the SCs of pairs VII and VIII (Fig. 11).Dispersed hybridization signals were observed inextended SCs, as seen for the chromosomes of pair VII.

The 5S rDNA locus, presented as two Fluoro-redhybridization signals, were intercalary on the shortarm of pair VI and corresponded to Giemsa C-bandsand DAPI/H-positive bands (Figs 12, 13).



Figures 2–10. Karyotype of Allium commutatum revealed by Giemsa C-banding, staining with CMA and silver staining.Fig. 2. NOR-bearing chromosomes of pairs VII and VIII have prominent C-bands at secondary constrictions (SCs) (arrow-heads). Chromosome pair VI has an intercalary C-band on the short arm (arrowheads). Note the heteromorphism ofbanding pattern between homologues of pairs VI and VII. The most prominent telomeric C-band on the satellites of thechromosome pair VIII (t) and weak centromeric C-band (c). Fig. 3. CMA-positive bands in NOR position (arrows) of chro-mosome pairs VII and VIII. Fig. 4. Subsequent silver staining of the cell shown in Fig. 3 revealing four Ag-NORs that cor-respond to CMA-positive bands. Note different size of homologous and nonhomologous Ag-NORs. Figs 5–9 Metaphaseplates with three and four Ag-NORs of equal and different sizes (arrowheads). The size of Ag-NORs corresponds to theextension of SCs. Fig. 10. Interphase cells with one to four nucleoli of equal or different size. Scale bar = 10 mm.

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DISCUSSION

The south-eastern Adriatic populations of A. commu-tatum studied here do not differ from other Mediter-ranean populations with regard to their chromosomenumber and karyotype characteristics (Garbari &Cela Renzoni, 1975; Maggini & Garbari, 1977;Mathew, 1996; Besendorfer et al., 1997). All have adiploid chromosome number (2n = 2x = 16) and theirkaryotypes have two NOR-bearing chromosome pairs,

pair VII (‘sativum’ type) and pair VIII (‘scorodopra-sum’ type).

In all the populations studied, the distribution of constitutive heterochromatin was the same as in the Palagruza population that we have already des-cribed (Besendorfer et al., 1997). The most prominentC-bands were those at the SCs of chromosomes VIIand VIII and at an intercalary position on pair VI. Incontrast to other Allium species, the chromosomes ofwhich are characterized by heavy telomeric C-blocks



11

12 13 14 15

Figures 11–15. Metaphase chromosomes of Allium commutatum after hybridization with 18S-26S and 5S rDNA probesand counterstaining with DAPI. Fig. 11. One step FISH on metaphase chromosomes revealing four green hybridizationsignals that correspond to 18S-26S rDNA genes located at the SCs of pairs VII (arrowheads) and VIII (arrows). Fig. 12.Red signal showing a 5S rDNA locus on the short arm of the chromosome pair VI (arrow). Figs 13–15. Chromosomes coun-terstained with DAPI showing DAPI-positive bands that are intercalary on the short arm of pair VI (Fig. 13; arrow), atSCs of pairs VII (Fig. 14; arrows) and VIII (Fig. 15; sc), at the centromere of all chromosomes (Figs 11, 15;c) and at thetelomere located on the satellite of pair VIII (Fig. 15;t).

on both chromosome arms (Vosa, 1976; Tardif & Morisset, 1991; Puizina & Papes, 1996), A. commuta-tum displayed faint telomeric C-bands. The exceptionwas the prominent telomeric C-band of the satellitedchromosome VIII. The C-banded karyotype of A. commutatum resembles that of A. porrum, a closelyrelated species in the A. ampeloprasum complex(Stack & Roelofs, 1996). Both species are character-ized by a small amount of constitutive heterochro-matin, most of which is associated with ribosomalgenes. Dimorphic C-bands, especially those that are

intercalary on chromosomes VI and at the SCs of chro-mosomes VII, is present in all A. commutatum popu-lations studied to date (Besendorfer et al., 1997 and this paper). Such dimorphism has been explainedin Drosophila as the result of unequal exchangesbetween sequences of heterochromatin (Fitch, Strausbaugh & Barret, 1990), that in the case of A.commutatum may have appeared as a consequence offrequent interstitial chiasmata (Besendorfer et al.,1997).

Characterization of NOR-associated heterochro-matin with different fluorochromes revealed that bothGC- and AT-rich sequences are probably intermingledin this chromosome region. Such heterochromaticorganization has already been described in A. flavum(Loidl, 1983), for Cestrum parqui, in which clusters ofsmall heterochromatic bands are scattered throughoutall chromosome arms (Berg & Greilhuber, 1992), andin the paracentromeric region of all chromosomes ofQuercus (Zoldos et al., 1999). DAPI and Hoechst stain-ing revealed intercalary AT-rich bands only in chro-mosome pair VI. However, a different banding patternachieved when DAPI is applied subsequent to FISHindicates that the removal of some chromatin com-pounds after denaturation allows better access of the fluorochrome to the heterochromatin. As a result,DAPI fluorescence appeared at the SCs, in telomeresof satellited chromosomes VIII and in centromeres of all chromosomes. Better DAPI staining following



Table 1. Number and position of active NORs (Ag-NORs) revealed by silver staining in six South Adriatic populations ofAllium commutatum. The maximum of four Ag-NORs located at SC of chromosome pairs VII and VIII as well as cells withone, two and three Ag-NORs are recorded

Number of metaphase plates in populations MetaphasesChromosome with Ag-NORs

Ag-NORs with Ag-NORs Palagruza Komiza Vis Bisevo Kastela Makarska Number (%)

1 1 (VII) 2 1 1 1 – 1 24 (10.5)1 (VIII) – 1 1 2 4 10

2 2 (VII) 1 1 2 – – – 61 (26.8)2 (VIII) – 2 1 1 5 41 (VII) + 1 (VIII) 7 7 7 8 8 7

3 1 (VII) + 2 (VIII) 4 9 4 9 10 4 71 (31.1)2 (VII) + 1 (VIII) 5 4 4 4 8 6

4 2 (VII) + 2 (VIII) 1 14 12 19 17 9 72 (31.6)

Total number of metaphase 20 39 32 44 52 41 228 (100)plates

Total number of Ag-NORs of 27 39 34 42 29 42 213 (46.61)chromosome VII

Total number of Ag-NORs of 22 44 33 53 39 53 244 (53.39)chromosome VIII

Table 2. Interphase cells with one, two, three and four nucleoli in six south Adriatic populations of Alliumcommutatum

Frequency of cells with 1–4 nuceoli (%)

Population 1 2 3 4

Palagruza 17.6 43.1 31.0 8.3Komiza 12.6 40.3 41.3 5.8Vis 11.0 44.0 31.0 14.0Bisevo 11.2 37.5 32.2 19.1Makarska 8.7 28.0 42.7 20.6Kastela 10.9 37.4 33.3 18.4Average 12.0 38.4 35.2 14.4

FISH has been observed in chromosomes of the species of Medicago (Calderini et al., 1996), Brassica(Maluszynska & Heslop-Harrison, 1993) and Quercusspecies (Zoldos et al., 1999).

In short, two types of heterochromatin were distin-guished after C- and fluorescent banding and FISH(Fig. 16): (a) NOR-associated heterochromatin of con-stitutive character composed of both GC- and AT-richintermingled DNA sequences; (b) the rest of the hete-rochromatin that is DAPI-positive after denaturation,centromeric (located on all chromosomes), telomeric(located on the satellite of chromosome VIII), andintercalary heterochromatin (located on chromosomeVI).

The Ag-NORs in studied A. commutatum popula-tions corresponded to C-bands, CMA/DAPI bands and18S-26S hybridization signals at the SCs of the chromosomes VII and VIII (Fig. 16). The maximumnumber of Ag-NORs at metaphase corresponded to themaximum number of nucleoli at interphase in all A.commutatum populations studied. However, the per-centage of Ag-NOR numbers at metaphase differedbetween populations although this percentage was not in positive correlation with the percentage ofnucleolus number seen in the interphases of the same populations. In the Komiza, Bisevo and Kastelapopulations the highest number of active NORs was three or four, while the predominant number ofnucleoli found at interphase in the same populationswas two or three. This indicates that homologous aswell as nonhomologous NORs prefer to associate whileactive.

Heteromorphism of Ag-NORs can be related to thenumber of ribosomal cistrons as shown in radioactive(Mellink et al., 1994) as well as nonradioactive in situhybridization experiments (Maluszynska & Heslop-Harrison, 1993). According to our FISH and silver-staining results it seems that Ag-NOR dimorphism inA. commutatum is due to the extension of SC and dif-ferent NOR activity, rather than the difference in thecopy number at homologous sites. Similar observa-tions were made in other species of genus Allium, e.g.A. sativum, A. cepa and A. fistulosum (Hizume et al.,1980; Sato, Hizume & Kawamura 1980; Schubert,1984). Comparison of the number of Ag-NORs and of 18S-26S rDNA sites indicates that some of thesesites are transcriptionally inactive in root-tip cells ofA. commutatum, despite the fact that they possessenough ribosomal cistrons to be detected by FISH. Ithas been suggested that the level of transcriptionalactivity may not depend on the number of ribosomalcistrons exclusively (Panzera et al., 1996; Zurita et al.,1997).

Even if some of the A. commutatum populationsanalysed originate from distant islands, no differenceswere found between them regarding quantity/distribution of constitutive and NOR-associated hete-rochromatin or NOR activity. The relatively recentgeographical isolation of the Adriatic islands (10000–30000 years) is obviously not sufficient for their populations to diverge karyologically.

The phylogenetic position of A. commutatum withinthe A. ampeloprasum complex is not completely clari-fied. It is known that polyploidization and naturalhybridization are actively taking place in the A.ampeloprasum complex, resulting in the continueddevelopment of new polyploid forms. Hybrids betweenA. ampeloprasum, A. commutatum and A. bourgeaui(the species comprising the A. ampeloprasum com-plex, together with A. atroviolaceum, sensu Bothmer,1974) have been observed on Crete (Bothmer, 1974).Moreover, Kik et al. (1997) have demonstrated thatthese species are interfertile with leek (A. porrum).Gross similarities at the level of chromosome mor-phology and amount of heterochromatin appear whenA. commutatum is compared to A. porrum (Stack &Roelofs, 1996), suggesting their close relationship. Kiket al. (1997) have, however, excluded the possibleancestral relationship between A. porrum and A. com-mutatum because of the lack of extra-mitochondrialDNA in the latter species. Our results, demonstratingdifferent numbers and positions of intercalary C-bands in the two species are in favour of this opinion.Karyological data, including modern banding tech-niques, and FISH mapping of rDNA loci for otherspecies from the A. ampeloprasum complex would,however, be required in the more detailed study of the species relationships. Lee, Do & Seo (1999)



Figure 16. Idiogram of A. commutatum chromosomes VI,VII and VIII. Characterization of heterochromatin revealedby banding techniques and position of 18S-26S and 5SrRNA genes by FISH. DAPI* = DAPI positive bands afterdenaturation in hybridization experiments: H* = Hoechstpositive bands after standard fluorochrome banding.

demonstrated that data on number/distribution of 5SrDNA loci are useful for such a purpose in the Alliumcomplex. Our results in A. commutatum and thoseobtained by Lee et al. (1999) in A. sativum show thattwo related and taxonomically close species, possess-ing very similar karyotypes, differ in the number andposition of 5S rDNA loci. Namely, there are three suchloci juxtaposed on the satellited chromosome bearinga major NOR locus in A. sativum and only one on thenon-NOR-bearing chromosome VI of A. commutatum.Data on number/position of 5S rDNA loci in otherspecies of A. ampeloprasum complex would thereforebe helpful in clarification of the phylogenetic relation-ship among them.

ACKNOWLEDGEMENTS

We thank Prof. Dieter Schweizer for providing probe3–3750–25S for in situ hybridization and Dr SonjaSiljak-Yakovlev from the University of Paris for usefulcomments and suggestions during the preparation ofthe manuscript. We also thank Ljiljana Buljubasic forcollecting the plant material. This work was supportedby the Ministry of Science and Technology, Republic ofCroatia (Project no. 119112).

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Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae

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