Heterochromatin differentiation in holocentric chromosomes of Rhynchospora (Cyperaceae)

Holocentric chromosomes of six species of Rhynchospora, R. ciliata, R. pubera, R. riparia and R. barbata (2n = 10), R. nervosa (2n = 30) and R. globosa (2n = 36), were stained with CMA3/DAPI fluorochromes or treated with C-banding and sequentially stained with Giemsa or CMA3/DAPI. Variability in banding pattern was found among the species studied. Heterochromatin was observed on terminal and interstitial chromosome regions, indicating that the holocentric chromosomes of Rhynchospora show a heterochromatin distribution pattern similar to those plant monocentric chromosomes.


INTRODUCTION
Heterochromatic sequence localization by banding techniques has contributed to our knowledge of chromosome organization, permitting discrimination between morphologically similar chromosomes, and aiding in establishing evolutionary relationships between closely related taxa. Few studies exist on the heterochromatin distribution pattern in holocentric chromosomes of plants. Initial research was done on the genus Luzula (Juncaceae) by Ray and Venketeswaram (1978) and Collet and Westerman (1984), who found interspersed among the euchromatin a variable number of C-bands, which fused during metaphase condensation. Thus, in prophase and prometaphase the band number was much larger than in metaphase. A similar situation was described for insect holocentric chromosomes (see Giles and Webb, 1972;Manicardi et al., 1991;Manicardi and Gautam, 1994).
Other work has demonstrated that holocentric chromosomes can exhibit defined heterochromatic blocks in telomeric regions (Panzera et al., 1992) as well as in telomeric and interstitial regions, e.g., in the Heteroptera Nezara viridula (Camacho et al., 1985). A similar case was reported by Kondo (1995, 1996) for several species of Drosera (Droseraceae), where interstitial C-bands were found in a few chromosomes and CMA3/ DAPI bands preferentially in telomeric regions.
The holocentric nature of Rhynchospora chromosomes has been discussed (Vanzela et al., 1996;Luceño et al., 1998). However, little information exists about the behavior and organization of the highly repetitive DNA in those chromosomes. The present article reports the heterochromatin distribution pattern in holocentric chromosomes of six Brazilian species of Rhynchospora, using three distinct chromosome banding methods.

MATERIAL AND METHODS
Six different species of the genus Rhynchospora (R. ciliata, R. pubera, R. riparia, R. barbata, R. nervosa and R. globosa) were studied. The voucher specimens collected in Northeastern and Southern Brazil are kept in the Universidade Federal de Pernambuco herbarium (Table I).
Root tips were pretreated with 2 mM 8-hydroxyquinolin for 24 h and further fixed in Carnoy (3:1, v/v) for 1-24 h. Tests were digested for 3 h in a mixture of 4% (v/ v) cellulase and 40% (v/v) pectinase, and squashed in a drop of 45% acetic acid. The coverslip was removed in liquid nitrogen and, after three days, the chromosomes were treated in three different ways. In the first treatment, R. ciliata and R. barbata samples were directly stained with a drop of 0.5 mg/ml CMA 3 in McIlvaine buffer, pH 7.0/distilled water (1:1) and 2.5 mM MgCl 2 for 1 h, washed in distilled water and stained with a drop of 2 µg/ml DAPI in McIlvaine buffer, pH 7.0, for 30 min, as described by Schweizer (1976). In the second, R. ciliata, R. barbata and R. nervosa samples were treated according to the Cbanding procedure (45% acetic acid at 60ºC for 10 min, 5% BaOH at room temperature for 10 min and 2xSSC, pH = 7.0, at 60ºC for 1 h and 20 min) and stained with 2% Giemsa, according to Schwarzacher et al. (1980). In the third treatment, R. ciliata, R. pubera, R. riparia and R. globosa samples were processed for C-banding, as mentioned above, and stained with CMA 3 /DAPI (Barros e Silva and . Materials stained with 2% Giemsa were mounted with Entellan (Merck) and those stained with fluorochromes were mounted in glycerin:McIlvaine buffer (1:1, v/v) and 2.5 mM MgCl 2 . Those stained with Giemsa were analyzed with an optical microscope, and photos were taken with Imagelink Heterochromatin differentiation in holocentric chromosomes of Rhynchospora (Cyperaceae) André L.L. Vanzela 1 and Marcelo Guerra 2 Abstract Holocentric chromosomes of six species of Rhynchospora, R. ciliata, R. pubera, R. riparia and R. barbata (2n = 10), R. nervosa (2n = 30) and R. globosa (2n = 36), were stained with CMA3/DAPI fluorochromes or treated with C-banding and sequentially stained with Giemsa or CMA3/DAPI. Variability in banding pattern was found among the species studied. Heterochromatin was observed on terminal and interstitial chromosome regions, indicating that the holocentric chromosomes of Rhynchospora show a heterochromatin distribution pattern similar to those plant monocentric chromosomes.
HQ 25 ASA Kodak film. Those stained with fluorochromes were examined using epifluorescence microscopy and photographed with T-MAX 400 ASA Kodak film.

RESULTS
Three different species were analyzed with C-banding: Rhynchospora ciliata (2n = 10) showed the highest number of terminal and interstitial blocks in almost all chromosome pairs (Figure 1a), R. barbata (2n = 10) exhibited a small pair of interstitial dots in each chromosome, in addition to terminal blocks (Figure 1b), and R. nervosa, with 2n = 30, presented heterochromatic blocks in some terminal and interstitial regions (Figure 1c).
The C-banding procedure, followed by CMA 3 /DAPI staining, also revealed variations in number and type of heterochromatic sequences among the four analyzed species. Rhynchospora pubera (2n = 10) exhibited three chromosome pairs with CMA 3 + terminal regions. One of these showed a larger and brighter signal, while the others Table I -Type and distribution of heterochromatin blocks on chromosomes of six species of Rhynchospora.

Species
Localities  showed weaker signals (Figure 2d and e). R. riparia, with 2n = 10, exhibited CMA 3 + blocks in four chromosome pairs. In two of these, blocks were only terminal, while in the two other pairs, bands appeared in terminal and interstitial positions (Figure 2f). DAPI bands were not observed in these two species. R. ciliata (Figure 2c) showed a band pattern similar to that obtained using C-banding and CMA 3 / DAPI staining. R. globosa was the only species that showed different patterns of CMA 3 and DAPI bands. DAPI + and CMA 3 + signals occurred in different terminal or subterminal regions on most of the chromosomes (Figure 2h). Besides, CMA 3 + bands always showed an appearance of small dots (Figure 2i), as in R. barbata. Interphasic nuclei showed randomly distributed CMA 3 + and DAPI + blocks (Figure 2f, g, i, and h, respectively).

DISCUSSION
The CMA 3 /DAPI staining, Giemsa C-banding and Cbanding followed by CMA 3 /DAPI staining revealed three different heterochromatin types in Rhynchospora holocentric chromosomes. The first type, characterized by CMA 3 + /DAPI 0 in R. ciliata, coincided with those obtained by Giemsa banding and was similar to that found in Drosera puchella and D. scorpioides holocentric chroa b c mosomes (Sheikh and Kondo, 1996). These regions could correspond to heterogeneous heterochromatin sites, where GC-rich segments are intercalated with AT-rich ones, as described by Guerra (1989) and Cuellar et al. (1996). The second type, CMA 3 + /DAPIbands present in most of the species, and the third type, CMA 3 -/DAPI + blocks, found only in R. globosa, seem to correspond to GC-rich and AT-rich DNA sequences, respectively (Schweizer, 1976).
Comparative band pattern analysis in these six species showed that size and location of heterochromatic segments were very variable. Heterochromatin may occur as large or small blocks (R. pubera and R. riparia), as few or multiple blocks along the chromosomes (R. barbata and R. globosa) and in terminal and/or interstitial regions. This heterochromatin segment distribution pattern resembles that observed in some plant groups (see Schweizer and Ehrendorfer, 1983). The data presented here differ from those observed for Luzula holocentric chromosomes (Ray and Venketeswaram, 1978;Collet and Westerman, 1984).
The variation observed in the holocentric chromosome banding patterns of Rhynchospora could be explained by the presence of different initiation and amplification heterochromatin sites (Peacock et al., 1981), possibly with dispersion by gene conversion or unequal crossing-over (Schweizer and Ehrendorfer, 1983) or by an equilocal dispersion mechanism similar to that proposed by Schweizer and Loidl (1987), for monocentric chromosomes. Terminal CMA 3 bands were observed in one or more chromosomes of all species studied. At least in R. ciliata and R. pubera, the CMA 3 blocks seem to correspond in position to rDNA sites described by Vanzela et al. (1998) through in situ hybridization. Therefore, some or all CMA 3 terminal bands observed in R. riparia, R. barbata and R. globosa could possibly be related to the NOR. Similar results were found for Drosera (Sheikh and Kondo, 1995).
These results suggest that no single typical pattern of heterochromatic segment distribution is present on holocentric chromosomes of Rhynchospora, in contrast with the conclusion presented by Collet and Westerman (1984) for Luzula holocentric chromosomes, i.e., "highlyrepeated DNA is not localized in single blocks, but is interspersed amongst the euchromatin". Band pattern variations may be more related to structural rearrangements, as in monocentric chromosomes (Deumling and Greilhuber, 1982;Schweizer and Ehrendorfer, 1983), rather than reflecting kinetochore organization on the chromosomes.

ACKNOWLEDGMENTS
The authors are grateful to CAPES, CNPq, BNB and FACEPE for financial support. Publication supported by FAPESP.