Genetics and Molecular Biology, 29, 1, 97-104 (2006) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br Research Article Karyotype differentiation of four Cestrum species (Solanaceae) based on the physical mapping of repetitive DNA Jéferson Nunes Fregonezi1, Thiago Fernandes1, José Marcelo Domingues Torezan2, Ana Odete S. Vieira2 and André Luís Laforga Vanzela1 1Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Biologia Geral, Londrina, PR, Brazil. 2Universidade Estadual de Londrina, Centro de Ciências Biológicas, Departamento de Biologia Animal e Vegetal, Londrina, PR, Brazil. Abstract We studied the karyotypes of four Brazilian Cestrum species (C. amictum, C. intermedium, C. sendtnerianum and C. strigilatum) using conventional Feulgen staining, C-Giemsa and C-CMA3/DAPI banding, induction of cold-sensitive regions (CSRs) and fluorescent in situ hybridization (FISH) with rDNA probes. We found that the karyotypes of all four species was 2n = 2x = 16, with, except for the eighth acrocentric pair, a predominance of meta- and submetacentric chromosomes and various heterochromatin classes. Heterochromatic types previously unreported 0 0 + - in Cestrum as neutral C-CMA3 /DAPI bands, CMA3 bands not associated with NORs, and C-Giemsa/CSR/DAPI bands were found. The heterochromatic blocks varied in size, number, position and composition. The 45S rDNA probe preferentially located in the terminal and subterminal regions of some chromosomes, while 5S rDNA appeared close to the centromere of the long arm of pair 8. These results suggest that karyotype differentiation can occur mainly due to changes in repetitive DNA, with little modification in the general composition of the conventionally stained karyotype. Key words: Cestrum, chromosome banding, CSRs, karyotype evolution, rDNA probes. Received: August 24, 2004; Accepted: May 31, 2005. Introduction such as those of the genera Solanum and Capsicum (2n=2x m Along with Vestia (Willd) and Sessea (Ruiz et Pav) = 24) exhibit small chromosomes (1.2-2.4 m for Solanum m the genus Cestrum (L.) is part of the tribe Cestreae (G. and 3.5-4.0 m for Capsicum) and constancy in the distri- Don) of the Cestroideae - Solanaceae). The genus Cestrum bution of meta- and submetacentric chromosomes (Ber- contains about 250 species distributed in the tropical and nardello and Anderson, 1990, Bernardello et al., 1994, subtropical regions of the Americas (D’Arcy, 1986) and in- Moscone, 1993). Nevertheless, the chromosomes of the cludes herbs, shrubs and trees up to 12 m in height. Mem- Cestreae are the largest in the family, reaching up to 14 mm bers of this genus are recognized by their actinomorphic and containing 1.36 pg of DNA per chromosome (Berg and flowers with tubular-infundibuliforme corolla, five sta- Greilhuber, 1992, 1993a,b, Sykorová et al., 2003a). mens attached to the corolla tube up to the middle or higher, Berg and Greilhuber (1992, 1993a,b) studied five bilocular ovary with berry-like fruits, few compressed species of Cestrum (C. aurantiacum, C. elegans, C. seeds and a straight embryo (Cronquist, 1988). The karyo- fasciculatum, C. parqui, and C. strigilatum) using several type of Cestrum species contains 2n = 2x = 16 chromo- banding techniques, and found four different heterochro- somes of similar size and shape, most of which are meta- or matic types: (i) large blocks unassociated with nucleolar or- submetacentric except for one subtelocentric pair (Berg and ganizing regions (NORs); (ii) numerous smaller blocks + Greilhuber, 1992, 1993a,b). In general, Solanaceae species forming intercalary, proximal or distal dots; (iii) CMA3 (chromomycin A3) bands associated with NORs; and (iv) DAPI+ (4’-6-Diamidino-2-phenylindole) bands associated Send correspondence to André L.L. Vanzela. Universidade Esta- with cold-sensitive regions (CSRs). The CSRs were first dual de Londrina, Centro de Ciências Biológicas, Departamento de Biologia Geral, Laboratório de Biodiversidade e Restauração de described by Dyer (1963) and at present have been reported Ecossistemas, Caixa Postal 6001, 86051-990 Londrina, PR, Brazil. in 11 genera of monocotyledons but only three dicotyle- E-mail: [email protected]. dons. 98 Fregonezi et al. Members of Solanaceae show large diversity in hete- squashed. The cover slips were removed after freezing in rochromatin accumulation and dispersion. Species of - liquid nitrogen and the preparations stained with 2% (w/v) Capsicum exhibited heterochromatic blocks in all the Giemsa and the cells photographed using Kodak Imagelink centromeres and secondary constrictions, besides a vari- HQ ASA 25 film. able number of large and small bands distributed along the For chromosomal banding, roots were digested in an chromosomes (Moscone et al., 1993). Similarly, Nicotiana enzyme solution composed of 4% cellulase and 40% species show localized DNA repetitive segments (micro- pectinase (both w/v) at 37 °C and dissected in a drop of 45% satellites, ribosomal DNAs and telomeric sequences), acetic acid. After removal of the cover slips, the slides were while other (virus-associated) segments are dispersed along aged for three days, and then incubated in 45% acetic acid, the chromosomes (Yoong Lim et al., 2000), and the litera- 5% barium hydroxide and 2xSSC, pH 7,0 (Schwarzacher et ture suggests that Cestrum can also follows this model. al., 1980, with modifications). The materials were stained During the research reported in this paper we com- in two different ways, either with 2% Giemsa or with the m pared the karyotypes of four Brazilian Cestrum species (C. fluorochromes 0.5 mg/mL CMA3 for 1.5 h and 2 g/mL amictum (Schlecht.), C. intermedium (Sendtn.), C. DAPI for 30 min. Slides stained with Giemsa were sendtnerianum (Mart. & Sendtn.) and C. strigilatum (Ruiz mounted in Entellan and those stained with the fluorochro- & Pav.)) using Feulgen staining and chromosomal markers mes mounted with a medium composed of glycerol/ produced by C-Giemsa and C-CMA3/DAPI banding, CSRs McIlvaine buffer (pH 7.0) 1:1 plus 2.5 mM MgCl2. The and fluorescence in situ hybridization (FISH) with 45S and cells were photographed using Kodak Imagelink HQ ASA 5S rDNA probes. The results are discussed considering the 25 and T-max ASA 100 film. possible mechanisms of dispersion and distribution of Fluorescent in situ hybridization (FISH) was per- heterochromatic segments and of rDNA cistrons in the formed according to the procedures described by Heslop- karyotypes of the four species. Harrison et al. (1991) and Cuadrado and Jouve (1994), with minor modifications. Slides were prepared as described for Materials and Methods banding. The wheat probes pTa71 containing the 45S Seeds of the four Cestrum species cited above (eight rDNA sequence (Gerlach and Bedbrook 1979) and pTa794 populations) were obtained from different localities and containing the 5S rDNA sequence (Gerlach and Dyer 1980) cultivated in tubes in the seedling nursery of the Laboratory were both labeled with biotin-14-dATP by nick translation m for Biodiversity and Restoration of Ecosystems (LABRE), and used for FISH. Each slide was treated with 30 Lofhy- Biology Science Centre, State University of Londrina, Pa- bridization mixture containing 100-150 ng of labeled probe m m raná, Brazil. Vouchers were deposited at the FUEL herbar- (4 L), 50% formamide (15 L), 50% polyethylene glycol ium (Table 1). (6 mL), 20xSSC (3 mL), 100 ng of calf thymus DNA (1 mL), Slides were prepared of root tips pre-treated with and 10% SDS (1 mL). The material was denatured at 90 °C 0.05% colchicine, fixed in Carnoy solution (ethanol/acetic for 10 min, and hybridization was performed overnight at acid 3:1, v:v), for up to 24 h and stored at -20 °C. Conven- 37 °C in a humidified chamber. Post-hybridization washes tional staining was carried out by the Fuelgen method. The were carried out in 2xSSC, 20% formamide in 0.1xSSC, cover slips were removed after freezing slides in liquid ni- 0.1xSSC and 4xSSC/0.2% Tween 20, all at 42 °C. The trogen and the slides permanently mounted in Entellan. To probes were detected with avidin-FITC conjugated and the obtain CSRs, roots were collected and pre-treated in Bristol post-detection baths were conducted in 4xSSC/0.2% nutritive solution (Bold, 1949) for 24 h at 0 °C and fixed in Tween 20 at room temperature. Slides were mounted in Carnoy solution. Roots were hydrolyzed in 1 M HCl, dis- 26 mL of a solution composed of antifade (12.5 mL) and sected in a drop of 45% (v/v) aqueous acetic acid and 50% glycerol in McIlvaine buffer (pH 7) with 2.5 mM Table 1 - Chromosome measurements and morphology of the Cestrum species and populations analyzed. CL = Length of large and small chromosome pairs; THL = Total length of haploid set. Cestrum species Natural location in Brazil CL (mm) THL Metacentric Submetacentric Acrocentric FUEL C. amictum Schlecht. Conquista farm, Rolândia, Paraná (PR) 6.9 to 9.8 70.2 1. 2. 3. 4. 6 and 7 5 8 30386 C. intermedium Sendtn. Mata dos Godoy State park, Londrina, PR 7.3 to 11.8 77.3 1. 2. 3. 5 and 7 4 and 6 8 31197 Mata Doralice reserve, Ibiporã, PR 30255 Rubi valley, Londrina, PR 31398 C. sendtnerianum Mart. Hermógenes de Freitas Leitão Filho ecological 6.4 to 8.7 63.4 1. 2. 3 and 4 5. 6 and 7 8 33213 park, Campinas, SP C. strigilatum Ruiz & Pav São Jerônimo native Brazilian reserve, São 7.2 to 8.6 63.3 1. 3. 4. 6 and 7 2. 5 and 8 - Jerônimo da Serra, PR 31953 Sapopema, PR 30984 Mata Doralice reserve, Ibiporã, PR 29682 Chromosomal physical mapping in Cestrum 99 m m m MgCl2 (12.5 L), plus 1 Lof50 g/mL propidium iodide. Photographs were taken using Kodak Proimage color ASA 100 film. In general, 15 specimens were analyzed for each of the eight populations and idiograms constructed based on the analyses of about 10 metaphases and/or pre-metaphases obtained from each treatment.