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Golczyk H., Hasterok R., Joachimiak A.J. 2005. FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea. Genome 48: 145-153.

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Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes was carried out in the complex permanent heterozygote Rhoeo spathacea. Telomere sites were exclusively terminal. All 10 25S rDNA loci were located
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  FISH-aimed karyotyping and characterization ofRenner complexes in permanent heterozygote  Rhoeo spathacea Hieronim Golczyk, Robert Hasterok, and Andrzej J. Joachimiak Abstract: Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes wascarried out in the complex permanent heterozygote Rhoeo spathacea . Telomere sites were exclusively terminal. All10 25S rDNA loci were located distally and appeared transcriptionally active after silver staining. Six distal and 2 in-terstitial 5S rDNA sites were detected; 2 of the distal sites strictly colocalized with 25S rDNA loci. The 2 intercalary5S rDNA loci occurred in short arms of 2 chromosomes that conjoined at meiosis. Chromosomes differed as to theamount of AT-rich centric heterochromatin, suggesting involvement of pericentromeric regions in translocations. Thepossibility of Robertsonian-like rearrangements was discussed. Double target FISH with ribosomal probes along withDAPI fluorescence gave the basis for full chromosome identification in mitosis. The 2 Renner complexes are structur-ally balanced, both having 5 25S and 4 5S rDNA sites. Centromere clustering, telomere association, a high number of NOR sites, and a strong tendency for formation of joint nucleoli contribute to the preservation of highly polarized Rablarrangement at interphase. These findings were discussed in relation to meiotic catenation in Rhoeo . Key words: chromosomes, complex heterozygotes, FISH, heterochromatin, interphase, meiotic multivalents, nucleolus,NOR, rDNA, Rhoeo , Renner complexes, translocations. Résumé : Une hybridation in situ en fluorescence (FISH) a été réalisée chez un hétérozygote permanent complexe du  Rhoeo spathacea à l’aide de sondes d’ADNr 25S, d’ADNr 5S et de séquences télomériques. Chacun des 10 locusd’ADNr 25S était situé en position distale et semblait activement transcrit après coloration à l’argent. Six locusd’ADNr 5S distaux ainsi que 2 locus intercalaires ont été détectés et 2 des locus distaux co-localisaient avec des locusd’ADNr 25S. Les 2 locus d’ADNr 5S intercalaires étaient situés sur le bras court de 2 chromosomes qui étaient jointslors de la méiose. Les chromosomes différaient quant à la quantité d’hétérochromatine centrique AT-riche ce qui sug-gère l’implication des régions péricentromériques dans des translocations. La possibilité de réarrangements robertson-niens est discutée. Des analyses FISH à double marquage, faisant appel à des sondes ribosomiques et au DAPI, ontpermis l’identification de tous les chromosomes lors de la mitose. Les 2 complexes de Renner sont équilibrés sur leplan structural, chacun possédant 5 sites d’ADNr 25S et 4 sites d’ADNr 5S. Le regroupement des centromères,l’association des télomères, le grand nombre de sites NOR et une forte tendance à former des nucléoles joints contri-buent au maintien d’un arrangement Rabl très polarisé au cours de l’interphase. Ces observations sont discutées en re-lation avec la concaténation méiotique chez le Rhoeo .  Mots clés : chromosomes, hétérozygotes complexes, FISH, hétérochromatine, interphase, multivalents méiotiques, nu-cléole, NOR, ADNr, Rhoeo , complexes de Renner, translocations.[Traduit par la Rédaction] Golczyk et al.153 Introduction Permanent translocation heterozygotes provide advanta-geous systems for the surveys of complex chromosome rear-rangements and their biological implications (Cleland 1972;Harte 1994). Nevertheless, the knowledge concerning theirkaryotype and mechanisms responsible for the meioticevents is mainly theoretical and largely incomplete. Furtherstudies should be focused on organisms expressing maxi-mum complex heterozygosity. Several heterozygotes of suchtype were described in plants (Stebbins 1951; Kenton et al.1987); however, many of them are not convenient for de- Genome 48 : 145–153(2005) doi: 10.1139/G04-093 © 2005 NRC Canada 145 Received 25 August 2004. Accepted 15 September 2004. Published on the NRC Research Press Web site at http://genome.nrc.ca on9 February 2005.Corresponding Editor: P.B. Moens. H. Golczyk 1 and A.J. Joachimiak. Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University,Grodzka 52, 31-044 Kraków, Poland. R. Hasterok. Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland. 1 Corresponding author (e-mail: h.golczyk@iphils.uj.edu.pl).  tailed cytogenetic analysis, e.g., the species Oenothera ,which possess very small chromosomes of almost identicalmorphology (Harte 1994).A spidewort, the boat lily Rhoeo spathacea Swartz(Stearn) is another textbook example of complex structuralheterozygosity in plants (Satterfield and Mertens 1972). Be-cause of its 12 large chromosomes and their mitotic appear-ance at meiosis, R. spathacea seems to be an especiallyfavourable candidate for chromosome research. In spite of this, an insight into its karyotype is far from satisfactory(Golczyk and Joachimiak 1999, 2003). In studies addressingmeiosis in Rhoeo , a stable relative position of chromosomeswithin multivalents was reported (Sax 1931; Lin and Pad-dock 1973). According to Sax (1931), there are 6 isobrachial(i) and 6 heterobrachial (h) chromosomes within thekaryotype of  R. spathacea , arranged within meiotic ringsand (or) chains in the following order: i h h i h h i h h i i i.In mitosis, some morphological chromosome types can bedistinguished statistically; in practice, it is rather impossibleto identify such types within a given metaphase plate with-out rather elaborated methodology (Golczyk and Joachimiak1999).According to Belling’s “segmental interchange theory”,which was srcinally based on the case of  Datura (Bellingand Blakeslee 1926), in some races of  Oenothera and in  Rhoeo , translocations had led to maximal heterozygosity,meaning that none of the chromosomes in the complementhad a fully homologous partner. The unusual mode of meio-sis with complete chromosome rings or chains arose as aconsequence. Heterozygosity in such plants is maintainedowing to the existence of the 2 multichromosomal super-linkage groups — Renner complexes (Flagg 1958; Cleland1972) — and owing to special breeding systems that elimi-nate homozygotes (Stebbins 1951).As far as we are aware, the method of fluorescence in situhybridization (FISH) has not yet been used in the analysis of   Rhoeo chromosomes. In this paper, we present advancedkaryotyping of  Rhoeo spathacea chromosomes with the useof 3 DNA probes: Arabidopsis -type telomeric arrays, 25SrDNA, and 5S rDNA supported by DAPI differential fluores-cence and silver staining. The abundance of 25S rDNA sites,their distal localization, and the reduced number of telomeresignals at interphase gives us the opportunity to make somesuggestions about the possible importance of nucleolus orga-nizing regions (NORs) for the nuclear chromosome layout,as well as for meiotic events in Rhoeo . Material and methods Plant material Three different Rhoeo clones were obtained from privatesources in Bulgaria, Costa Rica, and Poland. Plants weregrown in the greenhouse between 25 and 28 °C. Youngflower buds, approximately 2–4 mm in length, were isolatedfrom the inflorescences and fixed in 3:1 ethanol – glacialacetic acid. For the study on mitotic chromosomes, plantcuttings were initially grown in glass jars filled with tap wa-ter and wrapped with aluminum foil. When the roots reachedapproximately 3 cm they were cultured for 2 days in afreshly made Hoagland solution prepared according toDole ñ el et al. (1999). The roots were collected and pre-treated with a saturated solution of  α -bromonaphthalene for2 h, fixed in 3:1 ethanol – glacial acetic acid, and stored inthe fixative at –20 °C until use. Chromosome preparation For bright field analyses, fixed buds and roots werestained in 1% v  /  v acetic orcein or according to the Feulgenmethod. Dissected anthers and root tips were squashed in adrop of 45% v  /  v acetic acid. After freezing using the dry icemethod cover glasses were removed and the preparationswere air dried and made permanent in Entellan. For FISHexperiments, fixed root tips were enzymatically digested in amixture consisting of 40% v  /  v pectinase, 20% w  /  v cellulase,and 4% w  /  v cellulase Onozuka dissolved in citric acid –sodium citrate buffer (pH 4.8). Meiotic preparations weredone without any enzymatic treatment. After squashing (in45% v  /  v acetic acid) and drying, the preparations werestored at –20 °C until required. All preparations were exam-ined under phase contrast and those containing well-spreadmitotic chromosomes or meiotic rings and (or) chains com-pletely released from the callose wall were chosen as a sub-strate for FISH. Diploid metaphases, as well as sometetraploid metaphases in which the reproducibility of the hy-bridization pattern could be clearly seen (because each chro-mosome type is represented twice), were carefully analyzedwith respect to FISH signals. DNA probes and fluorescence in situ hybridization Three probes were used: HT100.3 Arabidopsis -typetelomeric repeats ((TTTAGGG) n ), 25S rDNA (Unfried andGruendler 1990), and 5S rDNA (clone pTa794) (Gerlach andDyer 1980). Both labelling and the FISH method followedthe protocols that were described in detail in Hasterok et al.(2001, 2002, 2004). The pictures were taken using aHamamatsu ORCA monochromatic CCD camera attached toa Zeiss Axioplan epifluorescence microscope. Artificial col-ors were applied using Wasabi software (Hamamatsu Pho-tonics, Germany, GmbH). The images were processeduniformly using Micrografx Picture Publisher software(Corel, Ottawa, Ont.), with the exception of very weak hy-bridization signals linked to some low copy number 25SrDNA loci, which required some over processing to be visu-alized. In some figures, DAPI fluorescence (blue) is shownin grey scale. Silver staining Root tips were pretreated in a saturated solution of  α -bromonaphtalene for 2 h and fixed overnight and refriger-ated in a mixture of 50% v  /  v ethanol – glacial acetic acidand 37% v  /  v formaldehyde, 18:1:1. Fixed root tips were en-zymatically digested for 30 min at 37 °C in a mixture con-sisting of 1% w  /  v pectinase and 1% w  /  v cellulase dissolvedin citric acid – sodium citrate buffer (pH 4.6). After squash-ing in 45% v  /  v acetic acid and freezing on dry ice, squasheswere air dried and stained according to Schwarzacher et al.(2004). Chromosome and karyotype analysis For the FISH/DAPI study, the best 15–18 meiotic ringsand (or) chains and 4–5 mitotic metaphase plates per clonewere selected and analyzed. For the analysis of NORs by sil- © 2005 NRC Canada 146 Genome Vol. 48, 2005  ver staining (Ag-NORs), the best 19–21 metaphases perclone were analyzed. The average lengths and arm ratios of individual chromosomes were calculated on the basis of measurements performed on digitally captured or scannedimages of chromosomes. The computer software packagesLucia G (Laboratory Imaging Ltd., Prague, Czech Republic)or Multiscan (Computer Scanning Systems Ltd., Warsaw,Poland) were used. Chromosome morphology was assessedin detail on the basis of arm ratio criteria proposed by Levanet al. (1964). Twelve Rhoeo chromosomes were numberedfrom 1 to 12 according to their relative position within mei-otic rings. This nomenclature is an adoption of the systemsrcinally proposed by Sax (1931) and later slightly modi-fied by Lin and Paddock (1973). In our system, chromosome1 corresponds to chromosome Aa, a counterpart of the chro-mosome 2 is chromosome aB, and so on. Results DAPI-FISH study and karyotyping Fluorescent in situ hybridization with the telomeric se-quence labelled all chromosome ends and appeared mainlyas twin dots (Fig. 1). No hybridization signals were detectedin the interstitial region of any chromosome. In the inter-phase nuclei, the telomeres lay in a typical Rabl orientation,opposite to the DAPI-positive collective chromocentresformed by pericentromeric heterochromatin (Fig. 2). Thenumber (from 5 to 14) and size of telomeric signals withinnuclei suggest telomere fusion occurring at interphase.The reproducibility of the FISH experiments with ribo-somal probes was ascertained not only in diploid (meioticand somatic) cells (Figs. 4 b and 4 c , 5 b and 5 c , and 6), but itwas especially evident in tetraploid ones, found within gen-erally diploid root meristems (Figs. 7 a– 7 b ). The unusual ex-cess of rDNA sites per diploid chromosome complementwas revealed. There were 8 5S rDNA sites on 7 chromo-somes (Figs. 4 b and 4 c , 6, 7 a , and 8) and 10 25S rDNA siteson 8 chromosomes (Figs. 5 b and 5 c , 7 b , and 8). Most of the5S rDNA hybridization sites were located distally, with theexception of those on the short arms of chromosomes 8 and9, which were interstitial (Figs. 4 b and 4 c , 6, and 7 a ). The 2other small 5S rDNA sites on chromosomes 11 and 12 areprobably also not telomere-adjacent (Figs. 6 and 7 a ). Thedistal regions of the shorter arms of all heterobrachial chro-mosomes harbored the major sites of 25S rDNA (Figs. 7 a and 7 b ). These arms conjoin at meiosis (Figs. 5 b and 5 c ).The 25S rDNA loci in the short arms of chromosomes 8and 9 were the largest and gave the strongest signals(Figs. 5 b and 5 c ). The minor 25S rDNA sites were localizedin the long arms of chromosomes 9, 10, and 11 (Fig. 8).These tiny loci generally displayed very weak fluorescence(Figs. 5 b and 5 c , 7 b ) and therefore were difficult to visual-ize, probably owing to low copy number of rDNA cistronswithin the locus.On the long arm of chromosomes 9 and 10, 5S rDNA and25S rDNA probes hybridized at the same position (Figs. 7 a and 7 b , 8). Both types of ribosomal DNA occur in the shortarms of chromosomes 8 and 9, but not in such a close prox-imity as in the long arm of chromosomes 9 and 10.All Rhoeo chromosomes, except the isobrachial chromo-some 4 and heterobrachial 3, showed brightly fluorescencingDAPI-positive segments of pericentric heterochromatin(Figs. 1, 4 a , 5 a , 6, 7 a and 7 b , Appendix A). These 2 chro-mosomes probably possess some minor AT-rich sites at thecentromeres, which in some preparations fluoresced dimly.Larger DAPI-positive signals were observed in chromo-somes 2, 8, 10, and 12, whereas very small signals were ob-served in chromosome 1 (Figs. 6, 7 a and 7 b ). In most cases,it was possible to make a distinction between adjoining iso-brachial (11–12) and heterobrachial (8–9) chromosomes.Chromosomes 12 and 8 showed larger DAPI-positive seg-ments in comparison to their “partners” (Figs. 4 c , 5 c , 6, 7 a and 7 b ). Also, chromosomes 2 and 6, virtually unrecogniz-able by rDNA distribution, differed by amount of AT-richpericentric heterochromatin (Fig. 6). Characterization of Renner complexes The double-target FISH with 5S rDNA and 25S rDNAprobes, combined with differential DAPI staining, allowedreliable identification of all Rhoeo chromosomes (Figs. 7 a and 7 b ). Different localization and intensity of FISH signalsand DAPI fluorescence may serve as excellent cytogeneticmarkers for individual chromosomes and enable more pre-cise characterization of Renner complexes in this species.These complexes ( α and β ) seem to be structurally balanced —both possess 3 major and 2 minor 25S rDNA sites, as wellas 4 5S rDNA sites (Fig. 9). Meiotic configuration of chromosomes All 3 analyzed clones of  Rhoeo were typical with respectto the sequence of their chromosomes at meiosis (Figs. 4 c ,5 c ), which was stable and more or less similar to that de-scribed by Sax (1931) and Lin and Paddock (1973). In allanalyzed rings and (or) chains, there were always 3 “pairs”of heterobrachial chromosomes conjoining by their shorterarms (chromosomes 2–3, 5–6, and 8–9), each of which wereflanked by isobrachial chromosomes (1, 4, 7, 10). The stablearrangement of the 10 chromosomes described above is verycharacteristic and consistent for Rhoeo , and that was themain reason to use it as a starting sequence in our chromo-some-classification system and karyograms (Figs. 4 c , 5 c , 8).The 2 less “metacentric” isobrachials (11 and 12), whichconjoined by their shorter arms, associate with the rest of thechromosomes. As was previously suggested (Golczyk andJoachimiak 1999), lengths of the arms of adjoining chromo-somes are similar, thus the superstucture at meiosis in Rhoeo fulfills the requirements of Bennett’s “natural karyotype”(Bennett 1984). The present study also confirms such a sup-position (Fig. 8). By morphology, 3 chromosome groupswere distinguished: large, nearly metacentric isobrachials(group I, chromosomes 1, 4, and 10); smaller isobrachials(group i, chromosomes 7, 11, and 12) with a marked armlength difference (but with arm ratios below 1.7); andheterobrachials (group h, chromosomes 2, 3, 5, 6, 8, and 9)with the highest arm ratios, generally above 1.7 (data notshown). The 2 large isobrachials — chromosomes 1 and 4— are the largest chromosomes within the Rhoeo karyotype.They both have one arm slightly longer than the other(Figs. 4 c , 5 c ) and average arm ratios of 1.17 and 1.15. Chro-mosome 10 is the smallest within group I and the only onein the karyotype possessing both arms of equal length (aver-age arm ratio = 1.01). The group of three large isobrachials © 2005 NRC Canada Golczyk et al. 147  © 2005 NRC Canada 148 Genome Vol. 48, 2005 Figs. 1–7. Chromosomes and nuclei of  Rhoeo spathacea , FISH, DAPI fluorescence, and orcein staining. Figs. 1 and 2. FISH withtelomeric sequences (red signals) performed on mitotic chromosomes and interphase nuclei, note a reduced number of telomere signalsat interphase. Fig. 3. Orceine stained somatic metaphase; I – large isobrachials; h – heterobrachials. Figs. 4 and 5. Meiotic ringsstained with DAPI (4 a and 5 a ), and after hybridization with 5S rDNA (4 b , red fluorescence) and 25S rDNA probe (5 b , green fluores-cence). Figs. 4 c and 5 c , chromosome drawings showing distribution of DAPI-positive segments (black) and rDNAs within meioticrings. Fig. 6. Diploid metaphase plate after FISH with 5S rDNA probe (red). Fig. 7. Tetraploid metaphase plate with two chromo-somes of each type. ( a ) 5S rDNA (red), ( b ) 25S rDNA (green). Arrows on Figs. 6, 7 a , and 7 b show interstitial 5S rDNA loci on chro-mosomes 8 and 9, whereas arrowheads show 5S rDNA sites on chromosomes 11 and 12, and triangles represent 25S rDNA sites thatcolocalize with 5S rDNA sites. Scale bar = 10 µ m.  is also distinguishable in mitotic preparations, but only chro-mosome 10 could be easily identified among them (Fig. 3).Chromosomes 7 and 11 are the smallest within theisobrachials, 12 is larger than chromosome 11 and hashigher arm ratio than its “partner”. Among the hetero-brachials (h), 2 chromosomes, 3 and 5, have the highest armratios in the karyotype — 2.75 and 2.38, respectively. Chro-mosome 5 was the longest among heterobrachials, and thusalso relatively easy to identify at mitosis (Fig. 3). Ag-NORs and nucleoli The analysis of chromosome spreads using silver staining(Figs. 10 and 11) revealed that most, probably all 10 25SrDNA loci (Fig. 8) were transcriptionally active. Althoughnot all Ag-NORs appeared in a given metaphase plate, theoverall distribution of silver deposits within the analyzedmaterial indicates the existence of 10 NORs (on 8 chromo-somes) per diploid cell. This is in disagreement with thehighest reproducible number of separate nucleoli, which was8 (Fig. 13). However, in Rhoeo , the number of nucleoli is apoor indicator of true NOR quantity owing to a strong ten-dency for nucleolar fusion in this species (Figs. 12 and 14).Some chromosomes differed by the size of silver-stained re-gions, suggesting differential activity of the NOR sites. Inthe majority of analyzed somatic metaphases, the 2 mostmassive silver deposits could be seen at the termini of 2 heterobrachial chromosomes (Figs. 10 a and 11). The 2largest NORs most often could be attributed to chromo-somes 8 and 9 (Figs. 10 a and 10 b ) and were traced to the2 largest 25S rDNA sites (Figs. 5 b and 5 c , 7 b ).Here we also focused on the associations between chro-mosomes and nucleoli to support the present FISH andsilver-staining studies. Such observations were carried outon late prophase chromosomes. The minimal and reproduc-ible number of chromosomes not attached to nucleoli was 3in all analyzed clones (Fig. 17), thus suggesting the exis-tence of 9 NOR chromosomes in Rhoeo . This does not cor-relate with 8 nucleolar chromosomes revealed by FISH(Fig. 8), and may suggest that there are more than 10 activerDNA clusters. Thus, it seems likely that in Rhoeo , the pres-ence of an additional, hardly detectable, rDNA site cannotbe excluded. © 2005 NRC Canada Golczyk et al. 149 Fig. 8. Ring-karyogram of  Rhoeo spathacea , based on fluorescence signals (DAPI/FISH) and chromosome measurements. Chromosomearm labelling after Sax (1931) and Lin and Paddock (1973). DAPI-positive heterochromatin shown in black. Fig. 9. The structure (5S and 25S rDNA sites) of the 2 Rennercomplexes in Rhoeo spathacea .
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