Morphological aspects of Passiflora edulis f. flavicarpa chromosomes using acridine orange banding and rdna-fish tools

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1 CARYOLOGIA Vol. 61, no. 2: , 2008 Morphological aspects of Passiflora edulis f. flavicarpa chromosomes using acridine orange banding and rdna-fish tools Praça Milene Miranda, Carlos Roberto Carvalho*, Francismar Correa Marcelino and Maria Andréia Correa MendonçA Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Universidade Federal de Viçosa, , Viçosa MG, Brazil. Abstract Passiflora edulis f. flavicarpa is considered the most important species of the genus Passiflora, mainly because of its botanical and commercial importance, as well as for crop breeding and genomic programs. However, its chromosome characterization has been conflicting on what concerns the number and localization of secondary constrictions (SC) and nucleolus organizer regions (NORs). In this context, conventional and molecular cytogenetic methodologies have been adapted to resolve these morphology problems. Cellular dissociation, airdrying techniques and Giemsa staining, acridine orange fluorochrome and fluorescent in situ hybridization (FISH) methodologies were used. We identified four secondary constrictions, in the subterminal portion of the long arm of chromosomes 1 and 8 and in the subterminal portion of short arm of chromosomes 2 and 7. Regions emitting yellowish fluorescence were observed on chromosomes 7 and 8 after acridine orange staining. The same pairs were evidenced with the rdna 18S probe used in the FISH technique, indicating that only two of the four secondary constrictions are related with NORs. Key words: Acridine orange, NOR, Passiflora edulis f. flavicarpa, passion fruit, rdna-fish, secondary constriction. INTRODUCTION Besides its agroeconomical and ecological importance, the genus Passiflora is also of great interest for cytogenetics, considering the amount of published works. However, the karyotype of Passiflora edulis Sims f. flavicarpa Deg. (2n=18) still presents conflicts concerning the morphology data of its chromosomes. Due to their importance for systematics, crop breeding and genomic programs of this plant, it becomes relevant that these conflicts be eliminated. The number and localization of secondary constrictions (SC), namely the morphological aspect of this chromosome region identified without any special banding, have been described differently. OLIVEIRA (1996) found one SC in chromosome 8, while SOARES-SCOTT (1998) showed two SC, one in chromosome 4 and another in chromosome 7. The latter author later related the presence of at least three SC, as cited by SOUZA et al. (2003). In contrast, CUCO et al. (2003) observed the presence of SC in chromosomes 8 and 9. * Corresponding author: phone: / ; fax: ; e- mail: ccarvalh@ufv.br Contradictory data have also been described in relation to the identification and chromosomic allocation of nucleolus organizer regions (NORs), meaning the region with active rdna genes identified by positive silver nitrate staining. Using this staining, MAYEDA (1997) detected NORs associated with the SC of chromosomes 8 and 9, although not specifying in which arm. With the advent of molecular cytogenetics, fluorescent in situ hybridization (FISH) has been the technique of choice for the specific localization of active or inactive rdna genes (LÓPEZ-LEÓN et al. 1999; SUMNER 2003). Using FISH in P. edulis f. flavicarpa chromosomes, MELO and GUERRA (2003) described differing morphological data regarding SC and NOR locations, in relation to previous authors. They observed sites of 45S rdna on the long arm of chromosomes 7 and 9, and 5S rdna localized on the long arm of chromosome 5. In addition to these methods, another procedure has been used to localize active or inactive NOR of plant chromosomes, based on indirect evidence yielded by the flanking heterochromatin associated with rdna genes (ALMEIDA and CAR- VALHO 2004). These authors used a hot denaturation pre-treatment and acridine orange fluorochrome to stain maize and pepper chromosomes

2 cytogenetic banding tools in passiflora 155 in a similar usage to the reverse banding (RFA) procedure described by VERMA and LUBS (1976) for human cytogenetics. Another improved cytogenetic tool has also been successfully applied for obtaining information from high quality plant chromosomes. As evidenced by CARVALHO and SARAIVA (1993; 1997) and ALMEIDA and CARVALHO (2004), the cellular dissociation and air-drying methods have demonstrated to be appropriate plant cytogenetic tools for excellent chromosome preparation for showing well-resolved morphological aspects. We applied the cellular dissociation and airdrying procedures, associated with acridine orange fluorochrome staining and the FISH techniques, for revising the NORs and SC number/position in the chromosomes of P. edulis f. flavicarpa. MATERIAL AND METHODS Plant material - P. edulis f. flavicarpa seeds (Passion fruit round yellow) were obtained from ISLA PAK Sementes Ltda., Porto Alegre, RS, Brazil ( The analyses were carried out at the Plant Cytogenetics and Cytometry Laboratory of the Federal University of Viçosa-UFV (Brazil). Probe labeling for the FISH technique was carried out at the Molecular Laboratory/ BIOAGRO-UFV. Pre-treatment and fixation - Seeds were germinated on distilled water in a Petri dish at 30 ºC. Seeds showing 1 cm roots were treated with a 3 µm amiprophos-methyl (APM, Nihon Bayer Agrochem K. K ) solution (DOLEZEL et al. 1999) for 16 h and 25 min at 4 ºC, washed with distilled water for 15 min, and then fixed in fresh cold methanol:acetic acid solution (3:1). The fixative was changed three times and the seeds were stored at -20 ºC. Slide preparations - Root-tips were washed three times with distilled water and placed in 1 ml of freshly prepared enzymatic solution consisting of 10 µl Flaxzyme (Novo Ferment TM ), which contains pectinolytic, cellulolytic and hemicellulolytic activities (CAIXETA and CARVALHO 2001), and 300 µl distilled water, for 1 h 30 min at 34 ºC. Roots were washed for 20 min with distilled water and fixed again at -20 ºC. Slides were prepared by meristematic cellular dissociation, air-dried and placed on a hot-plate (50 ºC) for 20 min (CAR- VALHO and SARAIVA 1993; 1997). Some slides were immediately stained with a 5% Giemsa (Merck ) solution in phosphate buffer (ph 6.8) for 4 min, washed twice in distilled water and air-dried. Acridine Orange (AO) staining - Slides were aged for days, then incubated in phosphate buffer (ph 4.7 at 85 ºC for 18 min) and stained with 0.01% (w/v) acridine orange for 15 min (AL- MEIDA and CARVALHO 2004). Fluorescent in situ hybridization - FISH was based on the methods of OSUJI et al. (1998). rdna probes containing the ribosomal 18S sequence (1100 pb) of Zea mays were cloned in pgem -T Vector (Promega ). The probes were labeled with reaction buffer by mixing 8 µl of fluor-12- dutp with 92 µl of 5x nucleotide buffer, by random priming, according to the recommendations of the Prime It kit (Stratagene ) manual. Slides with well-spread chromosomes of P. edulis f. flavicarpa were incubated with RNase (100 mg/ ml in 2xSSC) for 1 h in a moist chamber at 37 ºC, and then treated three times with 2x SSC (0.03 M sodium citrate and 0.3 M sodium chloride) in baths of 3 min. The material was dehydrated by 3 min incubation in each component of an alcohol series (50%, 75% and 100%). Samples of 15 ml of the probe were added to each hybridization area and the slide was covered with a coverslip and incubated for 3 min at 80 ºC, and overnight at 37 ºC. After hybridization, the slides were washed twice for 2 min in 2x SSC containing 50% (v/v) formamide, at 45 ºC; four times for 2 min in 2x SSC at room temperature; then once in PBS for 2 min. The preparation was counterstained with 25 ml propidium iodide (Sigma ) solution (1mg/ ml), washed in phosphate-buffered saline (PBS) for 1 min, and mounted with 15 ml Vectashield (Vector ). Image analysis - Images of chromosomes were captured with a CoolSNAP-Pro cf (Roper Scientific TM ) video camera of 12 bits on an Olympus TM BX-60 fluorescence microscope, with a 100x objective lens and a WB filter for analysis of acridine orange, and WG filters for FISH analysis. The frame was digitized using an Image Pro -Plus 4.5 software (Media Cybernetics TM ). Image analysis was performed on a Power Macintosh G4 computer, using the freely available ( ac.uk) Image SXM software (BARRETT 2002). This is a spin-off of the public domain image analysis application NIH Image, which was developed by RASBAND (1998). RESULTS Metaphase images were obtained showing a complete set of 18 chromosomes, well-spread and

3 156 praça, carvalho, correa marcelino and correa mendonça without overlapping on the same focal plane on the slide. The chromosome morphology showing well-defined primary and SC facilitated the characterization of the homologous pairs and, consequently, the karyogram assembly (Fig. 1 a). The analyses of pro- and metaphasic karyotypes stained with Giemsa revealed six pairs of metacentric (2-7) and three pairs of submetacentric (1, 8 and 9) chromosomes (morphometric data not shown). The SC were observed in the subterminal long arm of chromosomes 1 and 8 and subterminal short arm of chromosomes 2 and 7 (Figs. 1 b, c). The chromosomes stained with acridine orange showed four bright yellowish fluorescence regions, one localized on the terminal short arm of chromosome 7 and one on the terminal long arm of chromosome 8 (Fig. 2 a). Using 18S rdna probes in FISH, the target sequence was identified in interphasic nuclei showing four fluorescent spots (Fig. 2 b) and in two pairs of chromosomes showing four fluorescent signals. These signals were located on the distal short arm of chromosome 7 and on the distal long arm of chromosome 8 (Fig. 2 c). The chromosomes 7 and 8 were selected in order to highlight the morphological comparisons between the Giemsa staining, acridine orange fluorescence and FISH procedures (Fig. 2 d). DISCUSSION Root-tips treatment with APM, a phosphoric amide herbicide with significantly higher affinity to plant tubulins (DOLEZEL et al. 1999), enzymatic maceration, cellular dissociation and air-drying were considered important steps for providing slides showing high quality cytogenetic preparations. The APM treatment was considered adequate for accumulation of cells with a fine range of pro- and metaphasic condensation, for screening minor differences between chromosomes of the same class with apparent similar lengths (Figs. 1a c). The cellular fixation, maceration, dissociation and air-drying steps resulted in well-spread and morphologically preserved chromosomes. These methodologies, associated to the digital image analysis, contributed to discriminate subtle measurements between chromosomes. Similar results were obtained in Zea mays (CARVALHO and SARAIVA 1993; 1997), Capsicum annuum (ALMEIDA and CARVALHO 2004) and Aniba rosaeodora (CON- TIM et al. 2005). Four SC were found at the present work, localized in the subterminal long arm of chromosomes 1 and 8 and in the subterminal short arm of chromosomes 2 and 7 (Figs. 1 b, c). In contrast, using the conventional squashing technique, various authors differently described either the Fig. 1 Chromosomes of P. edulis f. flavicarpa (2n = 18 chromosomes) treated with APM 3 µm for 16 h and 25 min at 4 ºC and stained with Giemsa 5%. (a) Well-spread metaphase and (b) karyogram derived from prometaphasic and (c) metaphasic chromosomes. Notice the defined primary constrictions and the SC present in the distal long arm of chromosomes 1 and 8 and in the distal short arm of chromosomes 2 and 7 in both karyograms. Bar = 5 µm.

4 cytogenetic banding tools in passiflora 157 Fig. 2 Chromosomes obtained from meristematic cells of P. edulis f. flavicarpa, treated with APM 3 µm during 16 h and 25 min at 4 ºC, and interphasic nuclei. (a) Karyogram stained with 0.01% acridine orange. Notice the yellowish fluorescence signals on the distal short arm of chromosome 7 and on the terminal long arm of chromosome 8. FISH using the rdna 18S probe in (b) interphasic nuclei and (c) metaphasic chromosomes. Notice the presence of four yellowish fluorescent signals both in (b) nuclei and on (c) the distal arm of chromosomes 7 (short arm) and 8 (long arm). (d) Collection of chromosomes 7 and 8 stained with (G) Giemsa, (AO) acridine orange and using (F) FISH with 18S rdna labeled probe. Notice the correspondence between the SC with fluorescent signals in the short arm of chromosome 7 and long arm of chromosome 8. Bar = 5 µm. number or the localization of SC in P. edulis f. flavicarpa. OLIVEIRA (1996) found only one SC in chromosome 8, while MAYEDA (1997) related the presence of two pairs of chromosomes with satellites. SOARES-SCOTT (1998) showed a karyotype with two SC, one in chromosome 4 and another in chromosome 7, both in the long arm. In later works, SOARES-SCOTT related the presence of at least three SC, but did not specify their position (cited by SOUZA et al. 2003). CUCO et al. (2003) observed the presence of SC in chromosomes 8 and 9. According to MAYEDA (1997) differences on chromosome characterization may occur as a result of either the methods used or the quality of the sample preparation. In addition, the morphological identification of a particular SC in a chromosome without any special banding techniques depends on the level of chromatin condensation. The acridine orange staining, in accordance with the protocol used by ALMEIDA and CARVALHO (2004), showed a differential fluorescence pattern similar to that observed by these authors on chromosomes of pepper and maize. However, in these species, yellowish fluorescence bands were observed flanking the SC regions of the chromosomes 11 (pepper) and 6 (maize), while in passion fruit the fluorescent signals were observed on the terminal SC chromosomes 7 and 8, although not discriminating the flanking bands (Fig. 2 a). The differential fluorescence pattern in the SC region was obtained by treatment with buffer solution at ph 4.7 and 85 ºC for 18 min. Shifts for lower or higher values than these, in order to search for better resolution of the flanking regions, did not present satisfactory results. If the yellowish fluorescence of the SC of chromosomes 7 and 8 of passion fruit stained in a process similar to that of ALMEIDA and CARVALHO (2004), these regions also correspond to NOR-associated heterochromatin. Using FISH with 18S rdna probe two fluorescent signals were observed, one on the short arm of chromosome 7 and another on the long arm of chromosome 8 (Fig. 2 c). Similar results were obtained by MELO and GUERRA (2003), who iden-

5 158 praça, carvalho, correa marcelino and correa mendonça tified two sites of 45S rdna on the long arm of chromosomes 7 and 9 and one site of 5S rdna on the long arm of chromosome 5 in the karyotype of P. edulis f. flavicarpa. However, these data diverge mainly on what concerns the position of the two 45S rdna fluorescent signals corresponding to the SC, in short, whether one signal is present on the short or long arm of the chromosome 7, and whether the other signal is on chromosome 8 or 9. Such differences can be explained by the authors criteria, such as whether chromosome 7 had been oriented up or downwards, and if the positions of chromosomes 8 and 9 had also been exchanged in the karyogram. Although the origin of these punctual morphologic chromosome differences can be justified by interpretational problems related to the small size of the chromosomes, condensation degree or the cytogenetic methodology used, the differences between the 18S (present paper) and the 45S rdna (MELO and GUERRA 2003) probes used did not prevent the identification of SC that contain the genes. As shown by SUMNER (2003), the 45S unit includes the 18S sequence, since the NORs correspond to the sequences of repeated DNA encoding for 18S, 5.8S and 26S rrnas (BESEN- DORFER et al. 2002; SCHROEDER-REITER et al. 2006) and, cytologically, these regions are localized in SC in metaphasic chromosomes (BRASILEIRO-VI- DAL et al. 2003). According to BATTISTIN et al. (1999), all the NORs are localized in SC, but not all SC are NOR sites. With assistance of the techniques used in the present work, it was possible to identify four SC and two NOR sites. In conclusion, considering that the SC containing rdna sites could be identified by different staining methods, the Ag-NOR technique used by other authors could only identify the SC holding rdna genes that were active in nucleolus during preceding interphase. In this sense, some identification differences can be found between genotypes, populations and ecotypes. However, the acridine orange, which can be used for localization of heterochromatin associated with rdna genes, and rdna-fish methods allow the location of all rdna genes, active or non-active during interphase. Thus, if cases of intraspecific, genotypic or ecotypic differences are not present in these cytogenetic studies, our results suggest four SC (chromosomes 1, 2, 7 and 8) and two rdna sites, at the chromosomes 7 (short arm) and 8 (long arm) in P. edulis f. flavicarpa. Acknowledgements We thank the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) and the Conselho Nacional de Pesquisa (CNPq, Brazil) for the financial support. REFERENCES ALMEIDA P.M. and CARVALHO C.R., 2004 NOR-associated heterochromatin of pepper chromosomes stained with acridine orange. Caryologia, 57: BARRETT S.D., 2002 Software for scanning microscopy. Proceedings of the Royal Microscopy Society, 37: BATTISTIN A., BIONDO E. and COELHO L.G.M., 1999 Chromosomal characterization of three native and one cultivated species of Lathyrus L. in southern Brazil. Genetics and Molecular Biology, 22: BESENDORFER V., SAMARDZIJA M., ZOLDOS V., SOLIC M.E. and PAPES D., 2002 Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae). Botanical Journal of the Linnean Society, 139: BRASILEIRO-VIDAL A.C., CUADRADO A., BRAMME R.S.P., ZANATTA A.C., PRESTES A. M., MORAES-FERNANDES M.I.B. and GUERRA M., 2003 Chromosome characterization in Thinopyrum ponticum (Triticeae, Poaceae) using in situ hybridization with different DNA sequences. Genetics and Molecular Biology, 26: CAIXETA E.T. and CARVALHO C.R., 2001 An improved cytogenetic method for maize pachytene chromosomes. Cytologia, 66: CARVALHO C.R. and SARAIVA L.S., 1993 A new heterochromatin banding pattern revealed by modified HKG banding technique for maize chromosomes. Heredity, 70: and, 1997 High-resolution HKG-banding in maize mitotic chromosomes. Journal of Plant Research, 110: CONTIM L.A.S., CARVALHO C.R., MARTINS F.A. and FREI- TAS D.V., 2005 Nuclear DNA content and karyotype of Rosewood (Aniba rosaeodora). Genetics and Molecular Biology, 28: CUCO S.M., VIEIRA M.L.C. and AGUIAR-PERECIN M.L.R., 2003 Técnicas para a obtenção de preparações citológicas com alta freqüência de metáfases mitóticas em plantas: Passiflora (Passifloraceae) e Crotalaria (Leguminosae). Acta Botanica Brasilica, 17: 1-7. DOLEZEL J., CIHALIKOVA J., WEISEROVA J. and LUCRETTI S., 1999 Cell cycle synchronization in plant root meristems. Methods in Cell Science, 21: LÓPEZ-LEÓN M.D., CABRERO.J and CAMACHO J.P., 1999 Unusually high amount of inactive ribosomal DNA in the grasshopper Stauroderus scalaris. Chromosome Research, 7: MAYEDA L.Y., 1997 Estudo citogenético em dez táxons do gênero Passiflora L. (Passifloraceae). MS. Thesis, Universidade de São Paulo, Brazil.

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