CARYOLOGIA

*Corresponding author: phone/fax: ++48-81-4454610; e-mail: h.golczyk@wp.pl

Vol. 64, no. 3: 325-334, 2011

Cytogenetics of the permanent translocation heterozygote Rhoeo spathacea var. variegata. Implications for complex chromosome re-arrangements in Rhoeo

GOLCZYK* HIERONIM

Department of Molecular Biology, Institute of Biotechnology, John Paul II Catholic University of Lublin, Al. Krasnicka 102, 20-718 Lublin, Poland.

Abstract — The chromosomal morphology, arrangement of AT- and GC-rich chromatin, 5S and 45S rDNA, NORs, and centromere behaviour during somatic interphase and during meiotic prophase I were studied for the fi rst time in the variegated ring-forming Rhoeo variety – R. spathacea var. variegata. Fluorescence in situ hybridization (FISH) together with differential base-specifi c fl uorescence revealed the existence of the highly conserved karyotype in Rhoeo. It was shown that the two existing ring-forming varieties - R. spathacea variegata and R. discolor (common Rhoeo variety) share essentially the same arrangement of the studied cytogenetic landmarks within the chromosomal arms, together with the spread of the GC-rich DNA sequences (including rDNA) across all the twelve pericentromeres. They both also exhibit the same profound trend for centromere clustering in soma- and germ-line. The obtained results together with the existing literature served as a basis to summarize the cytogenetic knowledge on the karyotype structure in Rhoeo. A series of argumentations point-ing to the whole-arm translocations (WATs) as the rearrangements leading to meiotic catenation in Rhoeo was presented. A possible role of ancient inversions in the evolution of the PTH system in Rhoeo was discussed.

Key words: Centromeres, chromocenters, FISH, heterochromatin, interphase, meiotic prophase, multivalents, NORs, rDNA, permanent translocation heterozygosity (PTH), Rhoeo, translocations.

INTRODUCTION

Permanent translocation heterozygosity (PTH) in plants is a valuable tool for studying complex genome rearrangements. The textbook examples of this phenomenon are Oenothera (CLELAND 1972; HARTE 1984), Rhoeo (BELLING 1927; SAX 1931; GOLCZYK et al. 2005, 2010), Iso-toma (JAMES 1965). It has been widely assumed that during evolution of PTH extensive recipro-cal translocations have led to meiotic catenation, that is to rings and/or chains at meiosis, and to the severely restricted recombination (DARLING-TON 1931; CLELAND 1972; LIN and PADDOCK

1973; RAUWOLF et al. 2008). In a PTH organism there are two superlinkage groups, the so-called Renner complexes. Every second chromosome in the ring belongs to the same Renner complex and all the chromososmes of a given complex segregate as an unit because at metaphase I ki-netochores are alternately orientated, which al-lows every second chromosome to go to the same pole (CLELAND 1972; ÖSTERGREN 1951). The perpetuation of the heterozygous condition is accomplished by combining autogamy with special systems eliminating homozygotes, e.g. systems of recessive balanced lethals (STEBBINS 1950; CLELAND 1972; HOLSINGER and ELLSTRAND 1984; HARTE 1994, and the litererature therein). Unfortunatelly, the knowledge on the scenario of karyotype repatternings in PTH organisms in most cases is still unsatisfactory (discussed in: GOLCZYK et al. 2005).

The monotypic genus Rhoeo (2n=12), which was united with Tradescantia (HUNT 1986), was

GOLCZYK326

shown to be a good candidate for cytogenetic research on PTH (BELLING 1927; SAX 1931; GOLCZYK et al. 2005, 2008a; 2010). It encom-passes the two main ring-forming (ring of twelve chromosomes) varieties: R. spathacea (Syn. R. discolor) – the common variety with leaves uni-formly dark-green on the upper surface and purple on the lower surface, and R. spathacea variegata (Syn. R. spathacea vittata), – variegated variety, possessing leaves purple beneath, but variegated with longitudinal yellowish stripes on the otherwise dark-green upper side (BAKER and MERTENS 1975). All the essential cytogenetic data on PTH in Rhoeo was derived from the studies on the common Rhoeo variety (GOLCZYK et al. 2005, 2010, and the literature therein). As a result of advanced cytogenetic techniques ap-plied to meiotic and mitotic chromosomes of this variety, datailed standard karyotype based on chromosome arm measurements was cre-ated, and the chromosomal arrangements of such cyto-molecular landmarks like rDNA sites (45S and 5S rDNA), NORs (nucleolus organizer regions), AT/GC-rich genome fractions were uncovered (GOLCZYK et al. 2005, 2010). Previ-ous fi ndings have suggested the involvement of rRNA gene arrays in chromosome rearrange-ments in Rhoeo. 45S rDNA sites accompanied by CMA3+ (chromomycin A3) bands were detected within NOR-bearing distal chromo-some segments as well as within all the twelve transcriptionally silent pericentromeric regions (GOLCZYK et al. 2010). It was suggested that sub-telomeric 45S rDNA and pericentromeric chro-matin may have served as putative breakpoint regions, generating whole-arm translocations and/or whole-arm inversions. Paracentric inver-sions may have been ancient preliminary events seeding a part of 45S rDNA from distal chromo-some regions to pericentromeric ones (GOLCZYK et al. 2010). The persistence of the cytologi-cally well-defi ned chromatin domains (AT- and GC-rich chromatin, 45S rDNAs) within all the twelve pericentromeres indicates spreading and homogenization of their sequences during evo-lution of Rhoeo karyotype. A possible scenario may be that once a subtelomeric DNA sequence has been transferred to pericentromeric region by inversion, it could have later spread over the non-homologous pericentromeric regions due to intrinsic forces which bring proximal chromo-somal sites into close contact during interphase and meiotic prophase (GOLCZYK et al. 2008a, 2010). Indeed, unusually extensive centromere associations in somatic and meiotic cells de-

scribed in the common Rhoeo variety (COLEMAN 1941; NATARAJAN and NATARAJAN 1972; GOLCZYK et al. 2005; GOLCZYK 2011c) may form a nuclear framowork both for whole-arm translocations (WATs) and spreading/homogenisation of peri-centromeric DNA sequences (GOLCZYK et al. 2008a, 2010, and the literature therein). All the above fi ndings and proposals concern only one of the known PTH forms of Rhoeo, i.e. the com-mon Rhoeo variety (R. discolor). It is unknown if they could form the basis for a coherent view on the cytogenetic condition of the PTH in Rhoeo in general. The reason is that the arrangement of rDNA sites, NORs, AT- and GC-rich chroma-tin fractions was not studied in the second PTH Rhoeo form - R. spahacea variegata. Also nothing is known about centromere behaviour during interphase and meiotic prophase in R. spahacea variegata. Thus, the lacking knowledge on the karyotype structure and centromere behaviour in R. spathacea variegata is strongly needeed to assemble a gerenaralized view on PTH in Rhoeo.

The goal of the present investigation was to work out for the fi rst time the datailed standard karyotype (based on accurate chromosome arm measurements) and to study the chromosomal arrangement of rDNA sites (45S and 5S rDNA), NORs (nucleolus organizer regions), GC- and AT- rich chromatin fractions in R. spathacea var-iegata. To abtain this, FISH with ribosomal DNA probes (25S and 5S rDNA), Ag-staining, GC- and AT- specifi c differential fl uorescence was applied for the fi rst time to chromosomes of R. spathacea variegata. The present work aimed also at monitoring for the fi rst time the centromere behaviour during somatic interphase and early meiotic prophase in R. spathacea variegata. To achieve this, a differential fl uorescence technique revealing AT-rich chromatin fractions was used (see Metarials and Methods). The technique has recently proven to be useful in specifi cally detect-ing all the twelve pericentromeric regions in the common PTH variety of Rhoeo (GOLCZYK et al. 2010) and in monitoring centromere clustering in early prophase I (GOLCZYK 2011c).

MATERIAL AND METHODS

Plant material and chromosome preparations - Three clones of R. spathacea variegata were ob-tained from different private sources in Poland, Czech Republic and Hungary. All the plants were grown in the greenhouse at 25-28°C. For the studies on mitotic chromosomes, plant cut-

CYTOGENETICS OF RHOEO 327

tings and their roots were grown in tap water and were pretreated further as was already de-scribed (GOLCZYK et al. 2005). For base-specifi c fl uorescence and FISH young fl ower buds (ca. 5 buds per plant) and the pretreated root tips were fi xed in 3:1 ethanol – glacial acetic acid (AA). For silver staining AA fi xation was not applied – root tips were fi xed in a specially de-signed fi xative – FAA (50% alcohol, glacial acetic acid and 37% formaldehyde, 18:1:1), as was already described (GOLCZYK and JOACHI-MIAK 2003). Dissected anthers or root tips enzy-matically digested in 20% (v/v) pectinase, 20% (w/v) cellulase and 2% (w/v) cellulase Onozuka (dissolved in citric acid/sodium citrate buffer pH 4.6) were squashed in a drop of 45% acetic acid. After freezing using the dry-ice method,

cover slips were removed and the preparations were air-dried. For base-specifi c fl uorescence, only freshly made mitotic/meiotic prepara-tions were used. The preparations were stained with 4’-6-diamidino-2-phenyloindole (DAPI) or chromomycin A3 (CMA3) and respectively counterstained with actinomycin D (AMD) or distamycin A (DA) according to SCHWEIZER and AMBROS (1994). After CMA3/DA treatment, the preparations were further counterstained with DAPI. The best mitotic and meiotic prepara-tions after DAPI/AMD and CMA3/DA/DAPI treatments were subjected sequentially to FISH as described previously (GOLCZYK et al. 2010). For FISH, the best air-dried preparations were selected under phase contrast and treated with 0.1% pepsin solution in 0.01 N HCl for 15 min

TABLE 1 — Basic karyotypic parameters of Rhoeo spathacea variegata. Values are means. Standard deviations wit-hin brackets. 1-12, individual chromosomes; 1A-12A, individual chromosome arms; I, chromosome arm length; II, chromosome length; III, amount of AT-rich pericentromeric heterochromatin in each chromosome, [0.00] – AT-rich segments too small for cytological quantifi cation; IV, arm ratio; V, chromosome morphology; m – metacentric (arm ratio = 1.0-1.7), sm – submetacentric (arm ratio = 1.7 - 3.0), (i) – isobrachial, (h) – heterobrachial.

I II III IV V

11A 5.71 (0.39)

10.43 (0.63) 0.21 (0.11) 1.21 (0.10) m (i)1a 4.72 (0.38)

22a 5.27 (0.63)

7.75 (0.88) 0.92 (0.22) 2.13 (0.19) sm (h)2B 2.48 (0.31)

33B 2.36 (0.26)

7.69 (0.52) [0.00] 2.26 (0.35) sm (h)3b 5.33 (0.49)

44b 5.16 (0.56)

9.49 (0.73) [0.00] 1.19 (0.14) m (i)4C 4.33 (0.33)

55C 6.39 (0.56)

9.12 (0.72) 1.20 (0.25) 2.34 (0.27) sm (h)5c 2.73 (0.30)

66c 2.74 (0.40)

7.70 (0.72) 0.79 (0.15) 1.81 (0.26) sm (h)6D 4.96 (0.48)

77D 3.19 (0.31)

7.52 (0.55) 0.60 (0.20) 1.36 (0.13) m (i)7d 4.33 (0.33)

88d 4.88 (0.51)

7.69 (0.76) 0.95 (0.26) 1.74 (0.17) sm (h)8E 2.81 (0.34)

99E 2.70 (0.27)

7.71 (0.46) 0.52 (0.15) 1.86 (0.30) sm (h)9e 5.01 (0.46)

1010e 4.47 (0.47)

8.80 (0.70) 0.83 (0.21) 1.03 (0.03) m (i)10F 4.33 (0.33)

1111F 4.29 (0.64)

7.57 (0.92) 0.53 (0.19) 1.31 (0.31) m (i)11f 3.28 (0.54)

1212f 3.35 (0.41)

8.53 (0.83) 1.20 (0.12) 1.55 (0.17) m (i)12A 5.18 (0.54)

Total 100 100 7.75 (2.16) - -

GOLCZYK328

at 37°C, washed several times in 2 x SSC and dried through ethanol series. They were then post-fi xed in freshly depolymerized 4% (w/v) paraformaldehyde for 10 min and dehydrated in 70% and 100% ethanol for 5 min each, air-dried and stored at –20°C until required. For sil-ver staining, the dried preparations were further treated as described by HESLOP-HARRISON and SCHWARZACHER (2005).

Double-target fl uorescence in situ hybridiza-tion - The 5S rDNA-specifi c - the pTa794 clone (GERLACH and DYER 1980) was PCR-labeled with tetramethyl rhodamine-5-dUTP (Roche). The 2.3-kb Cla I fragment of the 25S rDNA of A. thaliana (UNFRIED and GRUENDLER 1990), labeled with digoxigenin-11-dUTP (DIG-11-dUTP) by nick translation (nick translation kit, Roche), was used for the detection of 45S rDNA sites. The standard high stringency FISH pro-tocol together with the signal detection system were as described previously (GOLCZYK et al. 2005 and literature therein). Additionally, low-stringency conditions recently described (GOL-CZYK et al. 2010) were applied to reexamine the chromosomal arrangement of any “weak” hy-bridization sites.

Image acquisition and processing, calcula-tions - The microphotographic documentation, image acquisition and processing were done as described previously (GOLCZYK et al. 2010). For measurements, karyogram constructing, and statistics, 21 mitotic metaphases (seven mitotic metaphases from each clone of R. spathacea var-iegata) after sequential DAPI/AMD-FISH treat-ments were used. Chromosome arm identifi ca-tion was carried out according to GOLCZYK et al. (2010). Chromosome measurements on digi-tally captured metaphases were carried out us-ing UTHSCSA ImageTool ver 3.0 (UTHSCSA ImageTool ver 3.0 (http://ddsdx.uthscsa.edu/dig/itdesc.html). The chromosome length and the amount of AT-rich heterochromatin were calculated as percentages of the total karyotype length, according to what was done previously (GOLCZYK et al. 2010).

RESULTS AND DISCUSSION

Chromosome morphology and organisation of cyto-molecular landmarks on chromosomes - It was shown in the common PTH Rhoeo variety (R. discolor) that there are six isobrachial (i) and six heterobrachial (h) chromosomes within its karyotype (SAX 1931). Following BELLING’S “seg-

mental interchange hypothesis” (BELLING 1927), the sequence of twelve meiotic chromosomes was coded: Aa(i)-aB(h)-Bb(h)-bC(i)-Cc(h)-cD(h)-Dd(i)-dE(h)-Ee(h)-eF(i)-Ff(i)-fA(i) – see SAX (1931), LIN and PADDOCK (1973), GOLCZYK et al. (2005, 2010). For convenience, to quickly refl ect the relative position of each chromosome wihin the ring, this meiotic arrangement was supported with number labellings by GOLCZYK et al. (2005, 2010), that is: 1(i)-2(h)-3(h)-4(i)-5(h)-6(h)-7(i)-8(h)-9(h)-10(i)-11(i)-12(i). This digit system can be easily combined with the let-ters, e.g. chromosome 12(fA), or chromosome arm 3b, etc. (see GOLCZYK et al. 2010). Thus, the α Renner complex consists of chromosomes 1(Aa), 3(Bb), 5(Cc), 7(Dd), 9(Ee), 11(fF), and the β complex encompassess the six remain-ing chromosomes: 2(aB), 4(bC), 6(cD), 8(dE), 10(eF), 12(fA) (FLAGG 1958; LIN and PADDOCK 1973; GOLCZYK et al. 2005, 2010; GOLCZYK 2011a). The perpetuation of the heterozygous α⋅β genotype within populations is most prob-ably governed by a genetic environment simi-lar to that of Oenothera lamarckiana (GRANT 1975), i.e. the system of recessive zygotic lethals (TSCHERMAK-WOESS 1947; CARNIEL 1960; WIM-BER 1968) establishing a balanced heterozygotic genotype L1l2/l1L2 in a repulsion phase linkage. The studied variegated variety of Rhoeo showed the twelve chromosomes in rings (Figs. 1A-B) or chains (Fig. 1C-F) at diakinesis/MI and the sequence of chromosomal types was: (i)-(h)-(h)-(i)-(h)-(h)-(i)-(h)-(h)-(i)-(i)-(i). Based on statisti-cal grounds (averaged measurements), it can be stated that in R. spathacea variegata, the terms “heterobrachial” and “isobrachial” conform ac-cordingly to submentacentric and metacentric types (Table 1). There are three large metacen-trics (chromosomes 1, 4, 10; arm ratio 1.0-1.2) and six submetacentrics (chromosomes: 2, 3, 5, 6, 8, 9; arm ratio 1.7-2.3). Within submetacentric chromosomes, chromosome 5 is the largest one (9,12 % of the karyotype). The three remaining metacentrics (chromosomes: 7, 11, 12) have both arms markedly uneven in length (1.3-1.5 arm ra-tio) . Measurements of each of the FISH-identi-fi ed chromosome arm in mitosis were the basis for constructing the standard karyotype of the variegata variety (Table 1, Fig. 2). The standard FISH with ribosomal probes on meiotic (Figs. 1C-D) and mitotic (Figs. 1G-H) chromosomes, as well as the base-specifi c differential fl uores-cence (Figs. 1K-L) revealed the chromosomal arrangement of basic cyto-molecular landmarks, that is 45S and 5S rDNA loci, AT-rich and GC-

CYTOGENETICS OF RHOEO 329

rich chromatin fractions to be essentially the same as in the common Rhoeo variety (see GOL-CZYK et al. 2005, 2010 for a comparison). There were no intravarietal differences in rDNA loci number and/or in chromosomal organization of GC/AT bands among R. spathacea var. variegata clones. Chromosomes 2, 3, 5, 6, 8, 9 have 45S loci localized distally on their shorter arms (Fig. 2). Chromosome 10 has two 45S rDNA loci each localized distally on one arm. Chromosome arms 9e and 10e have distal 5S and 45S rDNA loci co-localized. Each of the chromosomes 3 and 4 pos-sesses distal 5S rDNA locus on its arm b. Addi-tionally, interstitially located 5S rDNA loci could be detected on chromosomes 3, 4, 8, 9. Small 5S rDNA loci on the shorter f arms of chromo-somes 11 and 12, although located close to chro-mosome termini, actually are interstitially posi-tioned (Fig. 2). Interestingly, at high stringency weak pericentromeric 45S rDNA hybrydisation signals within meiotic chromosomes were fre-quently detected (Fig. 1E-F), whereas on mitotic chromosomes they were absent, which indicates that tiny 45S rDNA arrays are spread across all the twelve pericentromeres, and the differ-ence between meiotic and mitotic chromatin in 45S rDNA signal distribution may be due to a general difference in chromatin condensation between meiosis and mitosis, as was recently proposed for the common Rhoeo variety (GOL-CZYK et al. 2010). When lowering the stringency, 45S rDNA signals were clearly detectable within pericentromeric regions also on mitotic chromo-somes (Fig. 1G), as in the common Rhoeo variety (GOLCZYK et al. 2010). Silver staining revealed the transcriptional inactivity of the pericentro-meric 45S rDNA, and only distal localization of NORs (Fig. 1I). As many as ten separate nucleoli within interphase nuclei were consistently de-tected in all three clones (Fig. 1J), thus all the ten distally located 45S rDNA clusters are most likely NORs, as in the common PTH Rhoeo va-riety (GOLCZYK et al. 2005, 2010). Base-specifi c fl uorescence revealed that each pericentromeric region consists of the central AT-rich domain (Fig. 1K) and the associated one-two GC-rich segments (Fig. 1L), however some of the peri-

centromeric DAPI+ and CMA3+ bands were very

thin and their weak fl uorescence faded quickly, thus it was often not possible to document them all on microphotographs. Additionally, a few GC-rich segments which correspond to distal NORs were seen (Fig. 1L).

The AT-rich chromatin is restricted to peri-centromeric regions, which together with the calculated chromosomal amount of this chroma-tin fraction corresponds to what was reported for the common Rhoeo variety. For example, the total amount of AT-rich pericentromeric het-erochromatin in R. spathacea variagata – 7,75% (Table 1) is very close to 8.28% karyotypic con-tent of this fraction calculated for the common PTH variety of Rhoeo (GOLCZYK et al. 2010). In both the varieties chromosomes 3 and 4 have very little amount of AT-rich pericentromeric heterochromatin, frequently undetectable in the studied preparations. This is why these two chromosomes can be easily identifi ed even un-der applying simple DAPI-fl uorescence – they both frequently seem deprived of pericentro-meric heterochromatin while the rest of chrom-osmes possesses conspicious heterochromatic segments around centromeres (Fig. 1A).

Collective chromocenter/s in the somatic inter-phase and meiotic prophase I - The unusual ability of the centromeric regions of the common PTH Rhoeo variety to cluster into collective chromo-centers both in soma- and germ-line has been already well known (see Introduction). Present investigation has proven that centromeres exert the same strong trend towards organized clus-tering also in R. spathacea variegata (Fig. 1M-P). Root meristem nuclei showed clearly the pres-ence of the AT-rich collective chromocenters (arrows) occupying one nuclear hemisphere, whereas DAPI-negative nucleoli (arrowheads) were typically detected within the opposite hemi-sphere (Fig. 1M). Centromeres showed a clear tendency for clustering in early meiotic cells. In-terestingly, within young anthers, the prelepto-tene nuclei or very early leptotene nuclei, which did not show yet the presence of fi ne chromatin threads, often possessed one to several collec-

TABLE 2 — R. spathacea variegata. Length differences between arms that conjoin in the meiotic ring; Values are means. Standard deviations within brackets.

1a-2a 2B-3B 3b-4b 4C-5C 5c-6c 6D-7D 7d-8d 8E-9E 9e-10e 10F-11F 11f-12f 12A-1A Total

0.55(0.66)

0.12(0.08)

0.17(0.09)

2.06(0.59)

0.01(0.02)

1.77(0.42)

0.55(0.28)

0.11(0.10)

0.54(0.30)

0.04(0.03)

0.07(0.05)

0.53(0.24)

6.52(2.14)

Fig. 1 — R. spathacea variegata. Meiotic (A-F) and mitotic (G-I, K-L) chromosomes, and interphase nuclei (J). A, B – DAPI-stained meiotic ring (A) and its graphical interpretation (B), chromosomes numbered according to Golczyk et al. (2005, 2010), pericentromeric heterochromatin in B is drawn in black; C, D – meiotic chain after high-stringency FISH with the detected 25S rDNA loci (C, green signals) and 5S rDNA loci (D, red signals), arrows: interstitial 5S rDNA loci on chromosomes 3 and 4; E, F – meiotic chain after DAPI staining (E) and after standard FISH with 25S rDNA as a probe (F), arrowheads: pericentromeric 45S rDNA sites; G, H – 25S rDNA signals (green) seen on metaphase chromo-somes after applying low stringency FISH (G) and 5S rDNA loci (red) after reprobing the same preparation with 5S rDNA probe at high stringency (H), arrows: interstitial 5S rDNA loci on chromosomes 3 and 4, arrowheads: the dupli-cated interstitial locus on arms 8E and 9E; I, J – silver stained metaphase chromosomes (I) and nuclei (J), arrowheads: terminally located NORs; K – DAPI/AMD technique; L – CMA3/DA/DAPI technique, some of the pericentromeric CMA3

+ bands are out of focus; M – DAPI/AMD technique, root meristem nucleus; N – DAPI/AMD technique, preleptotene (left) and early leptotene nucleus with the fi ne chromatn threads beginning to appear (right); O – DAPI/AMD technique, pachytene nucleus with one collective chromocenter; P – DAPI/AMD technique, pachytene nucleus with three collective chromocenters, arrows: collective chromocenters, arrowheads: nucleoli. Scale bars = 5 µm.

CYTOGENETICS OF RHOEO 331

tive AT-rich chromocenters (Fig. 1N, left). This corroborates the recent report on the very early (preleptotene/leptotene) meiotic centromere clustering in the common Rhoeo variety (GOL-CZYK 2011c), but appears to be in disagreement with COLEMAN (1941), who observed twelve heteropycnotic bodies at leptotene, which later fused into collective chromocenter(s). As was suggested recently (GOLCZYK 2011c), the untypi-cal meiotic prophase of Rhoeo seems to comply with the concept of FUSSELL (1987), who viewed the meiotic bouquet as a possible prolongation of the premeiotic Rabl-polarisation. An ano-lagous situation seems to exists in allopolyploid wheats (NARANJO and CORRERDOR 2004). The meiotic centromere clustering was ubiquitous up till late pachytene. Nuclei with 1-3 collective chromocenters were most frequently seen within the whole fraction of the studied pachytene nu-clei (Figs. 1O-P). It emerges from the present study, that in both Rhoeo varieties the profound tendency of the AT-rich pericentromeric hetero-chromatin for self-adherence can unaubtedly be viewed as un universal feature of the PTH in Rhoeo (it exists in both PTH Rhoeo varieties). Similar heterochromatin stickiness (ectopic pair-ing) has been reported in Drosophila (MAYFIELD and ELLISON 1975), mouse (CERDA et al. 1999) and for AT-rich intercalary heterochromatin in Ornithogalum longibracteatum (PEDROSA et al. 2001). Furthermore, it could be now stated fi rmly, that similarily to Drosophila, in Rhoeo (both PTH varieties) the profound tendency of the pericentromeric heterochromatin of Rhoeo for self-adherence can reach its extreme when binding centric regions of all chromosomes within one large chromocenter (Fig. 1O). Inter-estingly, also in Oenothera centomere clustering

was shown to form a basis for a strong nuclear polarisation during meiotic prophase (GOLCZYK et al. 2008b)

Implications for chromosome rearrangements in Rhoeo - The present work together with the already existing data points to a high karyotypic and meiotic uniformity of the PTH in Rhoeo. Both PTH varieties possess essentially the same chromosomal arrangement of the cyto-molecular markers. They both are also characterized by the same ubiquitous centromere clustering in soma- and germ-line. It is likely, that the ring-forming Rhoeo is well the sole remnant of an ancient structural hybridity (STEBBINS 1950). As a result of the extensive comparison of both the PTH Rhoeo varieties, the below given suggestions and argumentations already mentioned when study-ing the common PTH Rhoeo variety (GOLCZYK et al. 2005, 2010, see also Introduction) could be now extended as the more generalized propos-als, valid for both PTH Rhoeo varieties.

In Rhoeo extensive rearrangements seem to have occurred in such a way as to preserve a high degree of length similarity between the arms that conjoin within the meiotic multiple (ring/chain). The simplest pathway to achieve this are WATs (whole arm translocations). Within most of the arm positions, homologous chromosome arms, in spite of some minor length mismatches, are visually well matched in length, and only at two positions (6D-7D and 4C-5C) they are vastly uneven in both Rhoeo varieties (Fig. 2, Table 2, compare with GOLCZYK et al. 2010). Impor-tantly, the submetacentric chromosomes, as well as the two chromosomes 11 and 12 conjoin by their shorter arms in both the two PTH varieties (Fig. 2, compare with GOLCZYK at al. 2010). In both Rhoeo varieties chromosomes differ as to

Fig. 2 — R. spathacea variegata. The measured chromosomes (1-12) arranged in the meiotic sequence. Chromosome arms are bent to graphically depict length relations.

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the amount of AT-rich pericentromeric hetero-chromatin (Table 1, compare with GOLCZYK at al. 2010). The total sum of length mismatches ranges around 6% of the genome (Table 2, com-pare with GOLCZYK et al. 2010), and although does not exceed the total amount of AT-rich pericentromeric heterochromatin (ca. 8%), is in fact close to it. This correlation strongly suggests that all the length mismatches in Rhoeo are likely the result of translocation breakpoints occurring within or near AT-rich centromeric heterochro-matin, but not exactly at the same distance from the centromeres. The chromosome arms 3b and 4b seem structurally homologous over their substantial lengths (40%), as can be inferred from the 5S rDNA pattern (Fig. 2). WATs are the mechanism which could easily explain the genome-wide homogenization of the pericen-tromeres by GC-rich DNA sequences, includ-ing rDNA, as discussed previoiusly (GOLCZYK at al. 2010, see also Introduction). WATs could be facilitated by the strong tendency for extensive centromere clustering in Rhoeo (see Introduc-tion), which was shown here to be a general fea-ture of this PTH system (Fig. 1M-P). The trans-location breakpoints usually tend to occur close or within heterochromatin (SCHUBERT 2007), which in both Rhoeo varieties is accumulated around centromeres (NATARAJAN and NATARA-JAN 1972; PETTENATI 1987; GOLCZYK et al. 2005, 2010). The suggested transfer of rDNA from subtelomeric to centromeric area in Rhoeo (see Introduction), could have predisposed centro-meres for fi ssion as introducing instability into these regions in the past (GOLCZYK at al. 2010, and the literature therein).

If WATs caused meiotic catenation in Rhoeo, then why are the translocated arms recombin-ing (sub)terminally? A possible explanation requiring further testing invokes ancient inver-sions (GOLCZYK et al. 2010). In general, these structural chromosomal changes have not been taken into account when considering the evolu-tion of PTH system in plants. The only struc-tural change which can effi ciently restrict re-combination, has no impact on chromosome morphology and is potentially able to cooperate with WATs in creating benefi cial linkages, is the paracentric inversion (see KIRKPATRICK 2010 for a review). Notably, in Oenothera the long-stand-ing dogma about a causal relationship between translocations and the restriction of meiotic re-combination, has been recently questioned by

us, i.e. evolutionary genetic factors other than translocations must have been the reason of the recombination shotdown in evening primroses (RAUWOLF et al. 2011). Inversions in combina-tion with translocations are now thought to be a frequent scenario leading to karyotype evolution (SCHUBERT 2007 and the literature therein). As it was proposed recently (GOLCZYK at al. 2010), the ancient initial events seeding 45S rDNA arrays into the pericentromeric area in Rhoeo could have been paracentric inversions involv-ing breaks within rDNA-bearing subtelomeric regions and other breakages – within pericentro-meric euchromatin-heterochromatin junctions. No such large-scale subtelomere-to-centromere inversion events could be traced in the closely related non-PTH Tradescantia virginiana, which similarly to Rhoeo possesses many distally locat-ed 45rDNA loci generating a strong CMA3

+ fl u-orescence. In contrast to Rhoeo, no 45S rDNA sites (after standard and low-stringency FISH) or any other generally defi ned GC-rich DNA clusters could be detected at pericentromeric positions in Tradescantia virginiana (GOLCZYK 2011b). Presumably, not only large, subtelo-mere-to-centromere inversions could have been involved in the evolution of the PTH in Rhoeo. Notably, in both PTH Rhoeo varieties the large 5S rDNA locus residing on each of 8E and 9E arms is a duplicated locus, consisting of two smaller loci separated by a short distance (Fig. 2, compare with GOLCZYK et al. 2010). The du-plication may have been due to small inversion with one of the breakpoints within the already existing 5S rDNA locus. Furthermore, each of the two interstitial 5S rDNA loci on chromo-some arms: 3b and 4b may have originated due to a middle-sized (ca. 40% of a given chromo-some arm) segmental inversion with one of the breakpoints within the telomere-adjacent major 5S rDNA locus. Based on the fi sh technique, mang small subterminal inversions were recen-tey proposed to have occurred in the closely re-lated Tradescantia virginiana (GOLCZYK 2011b). In general, inversions of different size and oc-curing throughout the chromosomal length have been very frequently described in Tradescantia species and hybrids (DARLINGTON 1929, 1937; SWANSON 1940; BHADURI 1942).

Acknowledgements — The study was partly sup-ported by grant N301 116 32/4008 from the Polish Ministry of Science and Higher Education.

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Received December 20th 2010; accepted November 2nd 2011