Ribosomal, Telomeric and Heterochromatin Sequences Localization in the Karyotype of Anemone Hortensis

ET AL .

Botanical Journal of the Linnean Society, 2006, 150, 177–186. With 14 figures

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

JELENA MLINAREC, DRAXEN A. PAPET and VITNJA BESENDORFER* Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 Department of Molecular Biology, Division of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia

Received February 2005; accepted for publication July 2005

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with empha- sis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2n = 2x = 16, common to all investigated populations, con- sisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by ﬂuorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Ara- bidopsis-type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Het- eromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis. Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 177–186.

ADDITIONAL KEYWORDS: ﬂuorescence in situ hybridization – ﬂuorochrome banding – karyotype analysis – meiosis – nucleolar organizer region – rRNA genes – telomeric repeats.

INTRODUCTION 1995; Ehrendorfer & Samuel, 2001; Schuettpelz et al., 2002). According to these data, A. hortensis is placed The genus Anemone (Ranunculaceae) comprises 70–90 in the Coronaria group together with all tuberous species of perennial, low-growing herbs. Two subgen- anemones from the Mediterranean region and south- era are recognized in the genus, which correlate with central USA and one disjunct species from South base chromosome number x = 7 for the subgenus America. Anemonidium and x = 8 for the subgenus Anemone. Species of Anemone were considered favourable Anemone hortensis L. as a tuberous Mediterranean plant material for cytogenetic studies because of species belonging to the subgenus Anemone is wide- chromosomal polymorphism, high levels of hetero- spread all along the Croatian Adriatic coast. Consid- chromatin and variation in its distribution, meiotic erable efforts, based on morphological and molecular chromosome behaviour as well as variation in total data, have been made in recent decades to improve our DNA content between species (Böchner, 1945; Rothfels knowledge of the phylogenetic relationships of the spe- et al., 1966; Baumberger, 1970; Cullis & Schweizer, cies of Anemone (Hoot, Reznicek & Palmer, 1994; Hoot, 1974; Marks & Schweizer, 1974). Cytogenetic analysis of anemones has mainly focused on determination of chromosome morphology and heterochromatin distri- *Corresponding author. E-mail: [email protected] bution using the Giemsa C-banding technique (Marks

178 J. MLINAREC ET AL.

& Schweizer, 1974). Cytogenetically, A. blanda has with the fluorochromes Chromomycin A3 (CMA) and been the most extensively explored species owing to its DAPI and FISH mapping of the ribosomal and telom- unique karyotype among anemones; levels and distri- eric sequences were employed. In addition, the behav- bution of heterochromatin have been well described iour of nucleolar organizer regions (NORs) in mitosis (Marks & Schweizer, 1974; Marks, 1976). Due to a and meiosis was analysed. large proportion (53–67%) of repetitive DNA found in the genome, efforts have been made to characterize MATERIAL AND METHODS satellite DNAs, as constitutive parts of heterochromatin, and determine their chromosomal position in PLANT MATERIAL A. blanda by fluorescence in situ hybridization (FISH) Plants forming part of the natural populations of the Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 (Cullis & Schweizer, 1974; Hagemann, Scheer & Adriatic islands of Hvar, Murter, Tisno, Krk and Vis, Schweizer, 1993). In A. blanda, a tandemly repetitive and mainland regions Makarska-Batko polje, Makar- sequence family (AbS1) is found to be located in all ska-Dugit, Makarska-Osejava and Starigrad-Paklen- 4,6-diamino-2-phenylindole (DAPI)-positive interca- ica were collected and potted in the Botanical Garden, lary bands and in the terminal DAPI-positive band Department of Botany, Faculty of Science, University of chromosome 3, whereas a dispersed repeated of Zagreb (Fig. 1). sequence (Hd), a part of the larger dorf-1 element that exhibits partial homology to the Lilium gypsy-type element del1, is preferentially associated with euchro- CHROMOSOME PREPARATION matic chromosome regions (Hagemann et al., 1993). Root tips were pretreated with 0.05% (w/v) colchicine Telomeres protect chromosome ends from degrada- (Sigma) at room temperature for 4 h, fixed in 3 : 1 (v/ tion and gradual shortening, maintain their structure v) ethanol/acetic acid at 4 °C for 24–48 h and stored in and regular segregation during the cell cycle, and 70% (v/v) ethanol. Floral buds collected in the field thereby contribute to genome stability. Telomeres were fixed directly in 3 : 1 (v/v) ethanol/acetic acid. consist of telomeric repeats, which are remarkably For karyotype observation, chromosomes were conserved in eukaryotes. The majority of plant stained with 1% (w/v) acetocarmine. Chromosome species analysed thus far possess Arabidopsis-type preparations used for fluorochrome and silver staining TTTAGGG telomeric repeats. However, a number of as well as in FISH experiments were prepared either exceptions have been described. Some species in the by softening in a mixture of 3% cellulase (Onozuka) order Asparagales (e.g. Allium) and in the family and 20% pectinase (Sigma) in 0.01 M citric buffer at Solanaceae (e.g. Cestrum) have been found to lack 37 °C for 45–55 min or by staining in 1% (w/v) aceto- Arabidopsis-type sequence repeats (Fuchs, Brandes & carmin, followed by maceration in 45% (v/v) acetic Schubert, 1995; Sykorová et al., 2003). Recently, inves- acid. A coverslip was removed from a slide by using tigations of chromosome ends in Aloe (Asphodelaceae), the dry-ice method (Sharma & Sharma, 1972) and air- Othocallis and Hyacinthella (Hyacinthaceae), all dried at room temperature for several days. belonging to the Asparagales, have revealed the pres- In the study of meiotic events, pollen mother cells ence of vertebrate-type TTAGGG telomeric repeats (PMCs) were extracted from anthers and slightly (Weiss & Scherthan, 2002; Puizina et al., 2003; Weiss- flamed in 45% acetic acid. Chromosomes in different Schneeweiss et al., 2004). As far as we are aware, stages of prophase I that were outside the cell walls there are no data regarding the characteristics of were selected for FISH experiments. telomeres in Ranunculaceae, including the genus Anemone. The most widely spread Mediterranean anemone in FLUOROCHROME AND SILVER STAINING Croatia is Anemone hortensis, which has not yet been Fluorochrome staining with CMA and DAPI was per- cytogenetically investigated. The only morphological formed according to the protocol of Kondo & Hizume study of A. hortensis in Croatia was by Radid (1987) on (1982) with slight modification. Slides were stained populations from Biokovo Mountain and the Makar- with 0.1 mg L−1 CMA for 15 min and counterstained ska region. Radid’s study showed that A. hortensis with 0.1% (v/v) methyl green or with 2 µg L−1 DAPI for appeared in a number of variants, such as A. hortensis 10 min. Silver staining was performed with colloidal var. heldreichii and A. hortensis var. stellata. The aim developer according to the method of Howell & Black here is to analyse the karyotype structure and genome (1980). organization of this species, in natural island and mainland populations including those described by Radid (1987), using morphometric data, the distribu- FLUORESCENCE IN SITU HYBRIDIZATION tion of heterochromatin, and the position of 5S and The position and number of 5S rDNA sites was 18S−5.8S−26S rDNA loci. For that purpose, staining determined by FISH, according to the method of

GENOME ORGANIZATION OF ANEMONE HORTENSIS 179

I. Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021

II.

III. IV. VII -IX. I. Krk II. Paklenica V. III. Murter VI. IV. Tisno V. Hvar VI. Vis VII. Makarska-Dugiš VIII. Makarska-Osejava IX. Makarska-Baško polje

Figure 1. Localities of investigated A. hortensis populations.

Heslop-Harrison et al. (1991), as modiﬁed by Ped- dUTP (Roche) using polymerase chain recation (PCR) rosa et al. (2001). Clone pTa794, containing the com- (Schwarzacher & Heslop-Harrison, 2000). Plant telo- plete 410-bp BamHI fragment of the 5S rRNA gene meric repeat probe (TTTAGGG)n was labelled with and the spacer region of wheat (Gerlach & Dyer, digoxigenin-11-dUTP (Roche) by PCR (Ijdo et al., 1980), was used as 5S rDNA probe. The probe was 1991). Slides were pretreated with RNase (10 mg ml−1, directly labelled with Cy3-dCTP (Amersham) by diluted 1 : 100 in 2× SSC) at 37 °C for 1 h, followed by − using a nick-translation kit according to the manu- Pepsin (Sigma) 10 mg ml 1, diluted 1 : 1000 in 0.01 N facturer’s protocol. HCl at 37 °C for 15 min in cases when FISH was per- The position and number of 18S rDNA sites was formed on pachytene. After digestion, spreads were determined using the 2.4-kb HindIII fragment of the ﬁxed with 4% (w/v) paraformaldehyde for 10 min, entire 18S rDNA from Cucurbita pepo, cloned into dehydrated through a graded series of ethanol and air- pUC19 as a probe (Torres-Ruiz & Hembelen, 1994). dried. Fifty microlitres of hybridization mixture con- The probe was labelled with Cy3-dCTP (Amersham) taining 60–80 ng of the probe DNA with 50% (v/v) for- by nick-translation as above or with digoxigenin-11- mamide, 10% dextran sulphate, 0.6% sodium dodecyl

© 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 177–186 180 J. MLINAREC ET AL. sulphate (SDS), 300 µg ml−1 salmon sperm and 2× SSC lowed. Chromosomes were numbered according to was applied to each slide and denaturated, together Marks & Schweizer (1974). with chromosomal DNA, at 75 °C for 7 min. Hybrid- ization was carried out at 37 °C overnight. After RESULTS hybridization, slides were washed stringently for 10 min in 30% formamide in 0.1× SSC when hybrid- Diploid chromosome complement (2n = 2x = 16) was ized with 18S rDNA probe or in 1× SSC without for- found in all nine investigated populations of Anemone mamide when hybridized with telomere probe. For hortensis. Morphological features of the chromosomes detection 18S rRNA genes, slides were pre-incubated are detailed in Table 1. The karyotype of Anemone con- in blocking buffer [3% bovine serum albumin (BSA) in sists of three acrocentrics (r = 4.41–4.84), chromo- 4× SSC/Tween20] at room temperature for 10 min, somes 1, 2 and 3; one meta-submetacentric (r = 1.60), Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 and for detection of telomeric repeats pre-incubation chromosome 4; and four metacentrics (r = 1.11–1.17), was carried out at 37 °C for 30 min. Incubation with designated chromosomes 5, 6, 7 and 8. Acrocentric anti-dig-ﬂuorescein isothiocyanate (FITC) (Roche) was chromosome 2 has a secondary constriction (SC) carried out at 37 °C for 1 h. After washing in 4× SSC/ (Fig. 2). Chromosome length varied from 6.17 to Tween 20 at 42 °C, three times every 10 min, chromo- 10.47 µm. The ratio of the longest to the shortest chro- somes were counterstained with DAPI (2 µg ml−1) and mosome pair was 1.7, and AsI was 63% (Table 1). slides were mounted in an anti-fade solution (Dako To determine heterochromatin distribution, chromo- Corp.). Signals were visualized and photographs were somes of A. hortensis were stained with CMA and captured on an Olympus BX51 microscope, equipped DAPI. Four CMA signals of similar intensity were with a highly sensitive digital camera (Olympus located on a satellite and SC of chromosome 2 and ter- DP70). Images were merged using Adobe Photoshop 6.0.

CHROMOSOME MEASUREMENT AND CONSTRUCTION OF IDEOGRAM Chromosomes from different populations were mea- sured on three well-spread metaphase plates. The morphometrical data include: (a) total chromosome length (TL), (b) relative length of each chromosome pair (chromosome length/TL × 100), (c) arm ratio of each chromosome (long arm/short arm), (d) ratio between the longest and the shortest chromosome pair and (e) the Asymmetry index (AsI% = long arm/ Figure 2. Karyotype of A. hortensis (2n = 2x = 16). Chro- TL × 100). For chromosome classiﬁcation the nomen- mosomes are ordered according to Marks & Schweizer clature of Levan, Fredga & Sandberg (1964) was fol- (1974). Chromosome 2 bears satellite.

Table 1. Morphometric data of Anemone hortensis chromosomes given as a total and relative chromosome length, the length of short and long chromosome arms and arm ratio (r). The morphology of the chromosomes was determined according to Levan et al. (1964): m = metacentric (r = 1–1.3), m-sm = metacentric–submetacentric (r = 1.3–1.7), a = acrocentric (r = 3.0–7.0). Total length of diploid complement is 131.56 µm with Asymmetric index 63% and ratio between the longest and shortest chromosome pair is 1.7

Chromosome Short arm Long arm Total length Relative length Arm Chromosome pair (µm) (µm) (µm) SD (%) SD ratio SD type

1 1.13 5.21 6.34 0.41 9.63 0.38 4.61 0.25 a 2 1.11 5.38 6.49 0.58 9.85 0.54 4.84 0.97 a 3 1.14 5.03 6.17 0.68 9.37 0.66 4.41 0.55 a 4 2.99 4.78 7.77 0.86 11.80 0.42 1.60 0.07 m-sm 5 4.07 4.77 8.84 0.89 13.42 0.24 1.17 0.05 m 6 4.53 5.18 9.71 0.86 14.74 0.24 1.14 0.06 m 7 4.68 5.40 10.08 0.97 15.30 0.38 1.15 0.07 m 8 4.97 5.50 10.47 0.91 15.89 0.42 1.11 0.08 m

© 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 177–186 GENOME ORGANIZATION OF ANEMONE HORTENSIS 181 minally on a short arm of chromosome 3 (Fig. 3). These (Fig. 9). In A. hortensis, 5S rDNA-associated hetero- four loci were also sites of hybridization of Cy3- chromatin was not positively stained either with DAPI labelled 18S rDNA probe (Fig. 8). These signals had or with CMA. The pattern of heterochromatin, rDNA similar intensities, suggesting similar copy number of loci and telomere sequence distribution is presented basic repeats. To determine whether all four loci were on the ideogram (Fig. 10). transcriptionally active we applied silver staining. During meiosis, pairing of chromosomes was regu- The maximum number of four nucleoli (data not lar. Diplotene and diakinesis clearly showed eight shown) corresponded to the two 18S−5.8–26S rDNA bivalents having chiasmata, localized proximally to loci, i.e. NORs, thus confirming that these two rDNA the centromere in both acrocentric and meta-submeta- loci can be active. In the interphase nuclei in which centric bivalents (Figs 13, 14). In acrocentric and some one nucleolus was forming, CMA signals were often meta-submetacentric bivalents interstitial and termi- Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 organized around the nucleolus, thus indicating the nal chiasmata were not present (Figs 13, 14), which association of NORs (data not shown). This was also resulted in formation of two and four armed bivalents confirmed by FISH in interphase nuclei, where a (Fig. 13). In pachytene and diplotene, staining with strong homogeneous 18S rDNA signal was observed CMA (data not shown) and FISH revealed fusion of (Fig. 11). heterologous 18S rDNA loci that formed one nucleolus Staining with DAPI showed distinctive intercalary (Fig. 12). and telomeric DAPI bands on the long arm of all acrocentrics. The three acrocentric chromosomes could be DISCUSSION distinguished according to the number and intensity of intercalary bands (Figs 4, 10). Satellite chromosome Adriatic populations of Anemone hortensis studied 2 had three intercalary DAPI bands in contrast to here have diploid chromosome number (2n = 2x = 16) chromosomes 1 and 3, which had two intercalary common to all anemones of the subgenus Anemone. DAPI bands. Metacentric chromosome 5 was charac- Two distinct karyotype classes were found in the Med- terized by a terminal DAPI band on the long arm. In iterranean Anemone species: one with three acrocen- all acrocentrics and metacentric chromosome 5, no trics and five meta-submetacentrics (A. coronaria, variability in DAPI banding pattern was noticed. On A. pavonina) and another with bimodal karyotype the contrary, the heteromorphism of the telomeric composed of four acrocentrics and four metacentrics DAPI band located on meta-submetacentric chromo- (A. blanda, A. palmata) (Marks & Schweizer, 1974; some pair 4 was found in all populations investigated. Medail et al., 2002). The karyotype of A. hortensis, Three different karyotypes could be distinguished comprising three acrocentrics and five meta-submeta- according to the presence or absence of telomeric centric chromosomes, is more similar to the karyo- DAPI bands: (1) without telomeric DAPI band (Fig. 6), types of two Coronaria group representatives, (2) telomeric DAPI band present in one chromosome of A. pavonina and A. coronaria. This similarity was fur- pair 4 (Fig. 4) and (3) telomeric DAPI band on both ther confirmed by the heterochromatin banding pat- homologues (Fig. 5). Interestingly, complete absence of tern in these three species (Marks & Schweizer, 1974; both CMA and DAPI bands was typical for the three this study). However, a variable telomeric DAPI band- largest chromosomes. ing pattern of chromosome pair 4 was noticed in The plant-type telomere probe efficiently hybridized A. hortensis populations investigated in this study. to the ends of all chromosomes, indicating the pres- To our knowledge, the position of ribosomal 5S and ence of TTTAGGG repeats in A. hortensis telomeres 18S−5.8S−26S rRNA genes has not yet been docu- (Fig. 7). Interstitial signals of telomere probe were not mented for any Anemone species. Our study showed observed. Different hybridization signal strength that the position and number of 18S−5.8S−26S rDNA between chromosomes suggested that chromosome sites in A. hortensis co-localized with four GC-rich ends had different telomeric repeat array length. heterochromatic regions as revealed by staining with Strong hybridization signals coincided with prominent CMA. Such a co-localization has been found in most terminal DAPI bands (Figs 7, 8). Faint hybridization dicot plant species (Guerra, 2000). In somatic inter- signals were observed at the ends of the three largest phase cells and meiotic cells of A. hortensis both 18S metacentric chromosomes, suggesting a lower copy rDNA loci were associating together, mostly forming number of the basic telomeric repeats. one nucleolus. In addition, a strong condensed 18S FISH with the 5S rDNA probe revealed four signals rDNA hybridization signal positioned near the nucle- on metaphase chromosomes that corresponded to two olus indicated that not all rRNA genes in tandem loci of 5S rRNA genes. One 5S rDNA locus was posi- array were active (Figs 11, 12). The association of tioned in the pericentromeric region on the short arm NORs is common in species where they are terminally of chromosome 4 and the other terminally on the short positioned (Zoldot et al., 1999; Hasterok et al., 2001), arm of chromosome 2 near the 18S rDNA locus which is also the case in A. hortensis. That is, owing to

© 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 177–186 182 J. MLINAREC ET AL. Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021

a high sequence similarity, terminally positioned centomeric sequences, which could lead to interlocus sequences have a greater likelihood of interacting exchange between 18S−5.8S−26S rRNA genes. Cronn with homologous and non-homologous chromosomes et al. (1996) suggested that genetic recombination, for (e.g. during end-to-end fusions) than interstitial or example, might be inﬂuenced by chromosomal loca-

Figures 3–10. Heterochromatin distribution and localization of ribosomal and telomeric sequences on metaphase chromosomes of A. hortensis. Scale bar = 10 µm. Fig. 3. Metaphase chromosomes after staining with CMA revealing four CMA signals on acrocentric chromosome pairs 2 and 3. Figs 4–6. Metaphase chromosomes after staining with DAPI showing intercalary DAPI bands on acrocentric chromosome pairs 1, 2 and 3 and telomeric DAPI band on long arms of all acrocentrics and terminally on meta-submetacentric chromosome pairs 4 and 5. Arrowheads indicate a variable terminal DAPI band on the meta-submetacentric chromosome pair 4. Fig. 7. FISH with digoxigenin-labelled plant telomere probe containing (TTTAGGG)n motifs (green signals) showing strong hybridization signals at the termini of all acrocetrics and one arm of the metacentric chromosome 5. Arrowheads indicate chromosome pair 4 bearing hybridization signals on one chromosome of the pair. Figs 8, 9. FISH with Cy3-labelled 18S rDNA and 5S rDNA probes. Arrowheads indicate four 18S− 5.8S−26S rDNA sites (Fig. 8) located on the short arm of acrocentric chromosome pairs 2 and 3 that correspond to CMA

signals (Fig. 3, arrowheads), and four 5S rDNA sites (Fig. 9) located on the short arm of chromosome pair 2 and in the Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 pericentromeric region of chromosome pair 4 (inset). Fig. 10. Ideogram of A. hortensis: 5S rDNA loci (red), 18S rDNA loci (orange), intercalary DAPI bands (light blue), telomeric FISH signals that correspond with DAPI bands (light green), telomeric dot signals (light green) and heteromorphic telomeric FISH/DAPI bands (dark green).

Figures 11–14. Behaviour of NORs in mitosis and meiosis of A. hortensis. Scale bar = 10 µm. Fig. 11. FISH with digoxigein-labelled 18S rDNA probe on an interphase nucleus revealing one large green hybridization signal that correspond to associated 18S−5.8S−26S rDNA sites. Fig. 12. FISH with digoxigein-labelled 18S rDNA probe on pachytene chromosomes, showing association of both homologous and non-homologous 18S−5.8S−26S rDNA sites. Figs 13, 14. Bivalents in diakinesis stained with CMA and counterstained with methyl green, and under the phase-contrast showing formation of two- and four-armed bivalents and interstitial and terminal chiasmata, respectively. Fig. 14. Two NOR-bearing bivalents associated together, with terminal ends forming one nucleolus. tion, which leads to sequence homogenization, as pos- 1999; Fulnedek et al., 2002), Nicotiana rustica sibly occurs in A. hortensis NOR sequences. Similar (Matyátek et al., 2003) and Gossypium (Cronn et al., situations have been reported in hybrid (allopolyploid) 1996). It will be interesting to analyse if homogeniza- species such as Nicotiana tabacum (Volkov et al., tion of rDNA occurs in other anemones, especially in

© 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 150, 177–186 184 J. MLINAREC ET AL. hybrid species such as A. fulgens, and whether it is could possibly explain the absence of banding pattern connected with the position and behaviour of NORs. In variation. contrast to the terminally located 18S−5.8S−26S The cytogenetic data presented in this and previous rDNA, 5S rDNA loci are positioned more internally, papers (Marks & Schweizer, 1974), based on chromo- near the centromere (chromosome 4) or NOR (chromosome morphology and heterochromatin distribution, some 2). The linkage pattern between 5S and 18S− agree with molecular data. Chloroplast and ribosomal 5.8S−26S rDNA sites has been observed in other restriction site variation presented by Hoot et al. angiosperms and gymnosperms (Kulak, Hasterok & (1994) and recently published results by Ehrendorfer Maluszynska, 2002; Siljak-Yakovlev et al., 2002; & Samuel (2001) based on atpB/rbcL spacer sequences Besendorfer et al., 2005). have also showed that A. hortensis and A. pavonina Distribution of telomeric sequences within a chro- are the most closely related species within the Coro- Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 mosome complement may be useful for the elucida- naria group. tion of the mechanism of karyotype evolution (Fuchs The unchangeable number and position of rDNA et al., 1995). Some telomeric or telomere-like DNA sites in A. hortensis indicated that this chromosome sequences have been reported in interstitial regions marker is suitable for phylogenetic analysis in anem- of chromosomes that are often thought to be rem- ones. The recent geographical isolation of the Adriatic nants of chromosomal rearrangements that have islands (10 000 years) has obviously not been suffi- occurred during genome evolution (Uchida et al., cient for populations to diverge in their rDNA site 2002; Tek & Jiang, 2004). Interstitial telomeric characteristics. Heteromorphy of the terminal DAPI sequences probably have arisen as a consequence of bands and FISH signals (chromosome pair 4) and the double-strand break repair by telomerase, or by chro- bimodal telomeric signal strength pattern could indi- mosome integration of extrachromosomal segments cate genome plasticity of this species in the sense or transposon-carrying telomeric sequences (Cherry of accumulation or reduction of heterochromatin & Blackburn, 1985; Murnane & Yu, 1993; Flint et al., sequences. 1994; Azzalin, Nergadze & Giulotto, 2001). In addi- In order to study genome evolution of anemones, tion, the interstitial position of telomeric sequences additional karyological data, including fluorochrome might indicate alteration of chromosome number banding, FISH mapping of rDNA loci as well as by chromosome fusion or inversion (Fuchs et al., characterization of repetitive sequences, should be 1995). Hybridization of Arabidopsis-type telomeric extended to other species. sequences (TTTAGGG)n, common to most plant species, occurred on the termini of A. hortensis chromo- ACKNOWLEDGEMENTS somes exclusively, and the absence of such sequences indicates that centromeric fusions or fissions, or We thank Sandro Bogdanovic, Petra Cigic, Iva associated changes in the chromosome number have Dobrovic, Duje Lisidic, Gordan Lukad, Marija Pand|a not been involved in the evolution of the Anemone and Marija-Edita Tolic for collecting material from karyotype. natural habitats, Andrea Pedrosa-Harand for valu- The intensity of telomeric signals varied signifi- able suggestions and helpful discussions and also cantly between the chromosome ends of A. hortensis Jasna Puizina for critical reading of the manuscript. chromosomes. Strong telomeric signals, found on the This work was supported by Grant no. 0119112 of longer arm of all acrocentric pairs, metacentric pair 5 the Ministry of Science, Education and Sport of the and heteromorhic chromosome 4, co-localized with Republic of Croatia and Bilateral Croatian–Austrian strong DAPI heterochromatic bands. Small dot-like project. telomeric bands, present on all other chromosome termini, did not have their DAPI counterparts. The dif- REFERENCES ference in telomeric hybridization signal strength Azzalin CM, Nergadze SG, Giulotto E. 2001. Human intra- reflects the unequal telomeric array length. The het- chromosomal telomeric-like repeats: sequence organization eromorphic terminal banding pattern of chromosome and mechanisms of origin. Chromosoma 110: 75–82. 4 could result from unequal crossing-over that leads to Baumberger H. 1970. Chromosomenzahlbestimmungen und an addition or deletion of telomeric repeats at chromo- Karyotypeanalysen bei den Gattungen Anemone, Hepatica some termini, producing homozygous and/or heterozy- und Pulsatilla. Berichte der Schweizerischen Botanischen gous individuals. Gesellschaft 80: 17–95. During meiosis, both acrocentric and meta-sub- Besendorfer V, Krajadic-Sokol I, Jelenic S, Puizina J, metacentric bivalents of A. hortensis formed proximal Mlinarec J, Sviben T, Papet D. 2005. Two classes of 5S chiasmata, which has also been described in A. blanda rDNA unit arrays of the silver fir, Abies alba Mill.: structure, (Marks, 1976). No chiasmata were seen to be involved localization and evolution. Theoretical and Applied Genetics in the banded regions of acrocentric bivalents and this 110: 730–741.

Böchner TW. 1945. Meiosis in Anemone apenina with special (TTAGGG)n generated by PCR. Nucleic Acids Research 19: reference to chiasmata localization. Hereditas 31: 221–231. 545. Cherry JM, Blackburn EH. 1985. The internally located Kondo T, Hizume M. 1982. Banding for the chromosomes of telomeric sequences in the germ-line chromosomes of Tet- Cryptomeria japonica D. Don. Journal of the Japanese For- rahymena are at the ends of transposon-like elements. Cell estry Society 64: 356–358. 43: 747–758. Kulak S, Hasterok R, Maluszynska J. 2002. Karyotyping of Cronn RC, Zhao X, Paterson AH, Wendel JF. 1996. Poly- Brassica amphidiploids using 5S and 25S rDNA as chromo- morphism and concerted evolution in a tandemly repeated some markers. Hereditas 136: 144–150. gene family: 5S ribosomal DNA in diploid and alloploid cot- Levan A, Fredga K, Sandberg A. 1964. Nomenclature for tons. Journal of Molecular Evolution 42: 685–705. centromeric position on chromosomes. Hereditas 52: 201–

Cullis CA, Schweizer D. 1974. Repetitious DNA in some 220. Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 Anemone species. Chromosoma 44: 417–421. Marks GE. 1976. Variation of Giemsa banding patterns in the Ehrendorfer F, Samuel R. 2001. Contributions to a molecu- chromosomes of Anemone blanda L. Chromosomes Today 5: lar phylogeny and systematics of Anemone and related 179–184. genera (Ranunculaceae-Anemoninae). Acta Phytotaxonom- Marks GE, Schweizer D. 1974. Giemsa banding: karyotype ica Sinica 39: 293–307. differences in some species of Anemone and in Hepatica nobi- Flint J, Craddock CF, Villegas A, Bentley DP, Williams lis. Chromosoma (Berlin) 44: 405–416. HJ, Galanello R, Cao A, Wood WG, Ayyub H, Higgs DR. Matyátek R, Lim KY, Kovarík A, Leitch AR. 2003. Riboso- 1994. Healing of broken human chromosomes by the addi- mal DNA evolution and gene conversion in Nicotiana rus- tion of the telomeric sequences. American Journal of Human tica. Heredity 91: 268–275. Genetics 55: 505–512. Medail F, Ziman S, Boscaiu M, Riera J, Lambrou M, Vela Fuchs J, Brandes A, Schubert I. 1995. Telomere sequence E, Dutton B, Ehrendorfer F. 2002. Comparative analysis localization and karyotype evolution in higher plants. Plant of biological and ecological differentiation of Anemone pal- Systematics and Evolution 196: 227–241. mata L. (Ranunculaceae) in the western Mediterranean Fulnedek J, Lim KY, Leitch AR, Kovar˘ ík A, Matyátek R. (France and Spain): an assessment of rarity and population 2002. Evolution and structure of 5S rDNA loci in allotetra- persistence. Botanical Journal of the Linnean Society 140: ploid Nicotiana tabacum and its putative parental species. 95–114. Heredity 88: 19–25. Murnane JP, Yu LC. 1993. Acquisition of telomere repeat Gerlach WL, Dyer TA. 1980. Sequence organization of the sequences by transfected DNA integrated at the site of a repeating units in the nucleolus of wheat which contain 5S chromosome break. Molecular and Cellular Biology 13: 977– rRNA genes. Nucleic Acids Research 8: 4851–4865. 983. Guerra M. 2000. Patterns of heterochromatin distribution in Pedrosa A, Jantsch MF, Moscone EA, Ambros PF, Sch- plant chromosomes. Genetics and Molecular Biology 23: weizer D. 2001. Characterization of pericentromeric and 1029–1041. sticky intercalary heterochromatin in Ornitogalum longi- Hagemann S, Scheer B, Schweizer D. 1993. Repetitive bracteatum (Hyacinthaceae). Chromosoma 110: 1224–1233. sequences in the genome of Anemone blanda: identiﬁcation Puizina J, Weiss-Schneeweiss H, Pedrosa-Harand A, of tandem arrays and dispersed repeats. Chromosoma 102: Kamenjarin J, Trinajstid I, Schweizer D. 2003. Karyo- 312–324. type analysis in Hyacinthella dalmatica (Hyacinthaceae) Hasterok R, Jenkins G, Langdon T, Jones RN, Maluszyn- reveals vertebrate-type telomere repeats at the chromosome ska J. 2001. Ribosomal DNA is an effective marker of Bras- ends. Genome 46: 1070–1076. sica chromosomes. Theoretical and Applied Genetics 103: Radic J. 1987. Genus Anemone L., section Anemone Tutin, 486–490. subsection Coronaria, subsect. nova in the region of Biokovo Heslop-Harrison JS, Schwarzacher T, Annamthawat- Mountain. Acta Biokovica IV: 73–96. Jonsson K, Leitch AR, Shi M, Leitch IJ. 1991. In situ Rothfels K, Sexsimth E, Heimburger M, Krause MO. hybridisation with automated chromosome denaturation 1966. Chromosome size and DNA content of species of Anem- techniques. Methods in Cell Molecular Biology 3: 109–116. one and related genera (Ranunculaceae). Chromosoma 20: Hoot SB. 1995. Phylogenetic relationships in Anemone 54–74. (Ranunculaceae) based on DNA restriction site variation and Schuettpelz E, Hoot SB, Samuel R, Ehrendorfer F. 2002. morphology. Plant Systematics and Evolution 9 (Suppl.): Multiple origins of Southern hemisphere Anemone (Ranun- 295–300. culaceae) based on plastid and nuclear sequence data. Plant Hoot SB, Reznicek AA, Palmer JD. 1994. Phylogenetic rela- Systematics and Evolution 231: 143–151. tionships in Anemone (Ranunculaceae) based on morphology Schwarzacher T, Heslop-Harrison JS. 2000. Practical in and chloroplast DNA. Systematic Botany 19: 169–200. situ hybridization. Oxford: Bios. Howell WM, Black DA. 1980. Controlled silver staining of Sharma AK, Sharma A. 1972. Chromosome techniques – the- nucleolus organizer regions with a protective colloidal devel- ory and practice. London: Butterworth (Publishers) Ltd. oper: a 1-step method. Experimentia 36: 1014–1015. Siljak-Yakovlev S, Cerbah M, Coulaud J, Stoian V, Ijdo JW, Wells RA, Baldini A, Reeders ST. 1991. Improved Brown SC, Zoldos V, Jelenic S, Papes D. 2002. Nuclear telomere detection using a telomere repeat probe DNA content, base composition, heterochromatin and rDNA

in Picea omorika and Picea abies. Theoretical and Applied Volkov RA, Borisjuk NV, Panchuk II, Schweizer D, Hem- Genetics 104: 505–512. belen V. 1999. Elimination and rearrangement of parental Sykorová E, Yoong Lim K, Fajkus J, Leitch AR. 2003. The rDNA in the allotetraploid Nicotiana tabacum. Molecular signature of the Cestrum genome suggests an evolutionary Biology and Evolution 16: 311–320.

response to the loss of (TTTAGGG)n telomeres. Chromosoma Weiss H, Scherthan H. 2002. Aloe spp.-plants with 112: 164–172. vertebrate-like telomeric sequences. Chromosome Research Tek AL, Jiang J. 2004. The centromeric regions of potato 10: 155–164. chromosomes contain megabase-sized tandem arrays of Weiss-Schneeweiss H, Riha K, Jang CG, Puizina J, telomere-similar sequences. Chromosoma 113: 77–83. Schertan H, Schweizer D. 2004. Chromosome termini of Torres-Ruiz RA, Hembelen V. 1994. Pattern and degree of the monocot plant Othocalis siberica are maintained by

methylation in ribosomal RNA genes of Cucurbita pepo L. telomerase, which speciﬁcally synthesizes vertebrate-type Downloaded from https://academic.oup.com/botlinnean/article/150/2/177/2420480 by guest on 30 September 2021 Plant Molecular Biology 26: 1167–1179. telomere sequences. Plant Journal 37: 484–493. Uchida W, Matsunaga S, Sugiyama R, Shibata F, Kazama Zoldot V, Papet D, Cerbah M, Panaud O, Besendorfer V, Y, Miyazawa Y, Hizume M, Kawano S. 2002. Distribution Siljak-Yakovlev S. 1999. Molecular-cytogenetic studies of of interstitial telomere-like repeats and their adjacent ribosomal genes and heterochromatin reveal conserved sequences in a dioecious plant, Silene latifolia. Chromosoma genome organization among 11 Quercus species. Theoretical 111: 313–320. and Applied Genetics 99: 969–977.