Somatic Chromosome Differentiation in Cucumis Melo L. and C

Chromosome Botany (2013) 8: 7-12 © Copyright 2013 by the International Society of Chromosome Botany Somatic chromosome differentiation in Cucumis melo L. and C. metuliferus E.Mey. ex Naudin Yoshikazu Hoshi1,6, Michiko Kido2, Kouhei Yagi3, Norikazu Tagashira4, Atsumi Morikawa5 and Katsuya Nagano1 1Department of Plant Science, School of Agriculture, Tokai University, Kawayo, Minamiaso-Mura, Aso-Gun, Kumamoto 869-1404, Japan; 2Graduate School of Agriculture, Tokai University; Tokai University, Kawayo, Minamiaso-Mura, Aso-Gun, Kumamoto 869-1404, Japan; 3Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787 Warszawa, Poland; 4Department of Liberal Arts, Faculty of Liberal Arts, Hiroshima Jogakuin University, 4-13-1 Ushita Higashi, Higashi-Ku, Hiroshima-City, Hiroshima 732-0063, Japan; 5Agricultural Training Station, School of Agriculture, Tokai University, Kawayo, Minamiaso-Mura, Aso-Gun, Kumamoto 869-1404, Japan 6Author for correspondence: ([email protected]) Received January 25, 2013; accepted February 22, 2013 ABSTRACT. Fluorescent banding with chromomycin A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI), and fluorescence in situ hybridization (FISH) were employed to investigate the chromosome differentiation in C. melo and C. metuliferus. The chromosome number of 2n=24 was counted in the two species. Average chromosome lengths, total chromosome lengths and ranges in chromosome lengths of the two species were very similar to each other. In spite of the karyomorphological similarity, fluorescent chromosome banding pattern was quite different in each other. Unlike the C. melo, C. metuliferus had unexpected chromosome- specific CMA and DAPI bands, especially in prometaphase. In FISH, two 5S rDNA and four 45S rDNA signals were detected at the ends of chromosomes of C. melo, while two 5S rDNA and two 45S rDNA signals were detected at the ends of chromosomes of C. metuliferus. KEYWORDS: Chromosome differentiation, Cucumis melo, Cucumis metuliferus Cucumis consists of 32 species, including a few have any basically detailed chromosome information. economically important species. Melon (C. melo L., In this paper, fluorescent banding with chromomycin 2n=2x=24) is one of the most important vegetable crop in A3 (CMA) and 4’,6-diamidino-2-phenylindole (DAPI), world production, the same as cucumber (C. sativus L., and fluorescence in situ hybridization (FISH) were 2n=2x=14). Except for cucumber, all Cucumis species employed to investigate the chromosome differentiation have somatic chromosome number of 2n=24 with the in C. melo and C. metuliferus. basic chromosome number of x=12. Because chromosome complements of the most species are similar to those of C. MATERIALS AND METHODS melo, many attempts of pollination-crossing with C. melo Plant materials The cultivars of two Cucumis species, C. have been made to introduce several genetic characters melo L. cv. ‘New melon’ and C. metuliferus E. Mey. ex from the other Cucumis species, especially African honed Naudin. were used in this study. These seeds are stocked cucumber (C. metuliferus E. Mey. ex Naudin., 2n=2x=24), in the Department of Plant Science, School of Agriculture, also known as ‘Kiwano’ in New Zealand. Tokai University. Cucumis metuliferus is the third important crop in the genus, and has several attractive natures with the Chromosome preparation Mitotic chromosome slides tolerances such as disease, insects and nematodes (Norton were prepared using the enzymatic digestion method. For and Granberry 1980). Although the transfer of these fluorescent staining and fluorescencein situ hybridization valuable genes from C. metuliferus to C. melo would be (FISH), slides were prepared according to a previous desirable, the cross pollination to set fruit of F1 hybrid method (Hoshi et al. 2008). Root tips for chromosome between them is usually quite difficult (George 1970; preparation were collected from seedlings. They were Deakin et al. 1971). Despite the hardship for creating the treated with 0.05% colchicine at 18°C for 2 h and fixed in interspecific hybrid, Norton and Granberry (1980) 3:1 ethanol-acetic acid for 1 h, and washed with distilled reported that the hybridization between C. melo and C. water for 1 h at 4°C. Those roots were cut into ca. 2 mm metuliferus gave occasionally normal fruit production long and treated with 4% Cellulase ‘Onozuka’ R10 with viable seeds. This result is notable success for (Yaklut) and 2% Pectolyase Y-23 (Kikkoman) at pH 4.8, developing the C. melo improvement by use of C. 37°C for 1 h. Then, they were washed with distilled water metuliferus. Until now, however, C. metuliferus did not at 4°C for 1 h, and chopped on a glass slide with 3:1 8 HOSHI ET AL. ethanol-acetic acid. 10 min twice. Then, the slides were air dried after ethanol series (30%, 70% and 99.5% (v/v)) for 10 min, Differential fluorescent staining with CMA and DAPI For respectively. The slides were then mounted in Vectashield fluorescent differential staining with chromomycin 3A mounting medium (Vector Lab.) containing 0.5 µg/ml (CMA) and 4’,6-diamidino-2-phenylindole (DAPI), the DAPI. After the treatment, they were irradiated with U method of Pląder et al. (1998) was followed with slight filter cassette and fluoresced blue for obtaining counter modifications. The air-dried slides were treated with stained images, were irradiated with IG filter cassette for McIlvaine’s buffer (pH 7.0) for 30 min, 0.1 mg/ml CMA detecting fluorescent color of rhodamine, and were in McIlvaine’s buffer supplemented with 5 mM MgSO4 irradiated with IB filter cassette for detecting fluorescent for 10 min at room temperature under dark condition, and color of Alexafluora 488. Photographs were taken with then they were mounted by glycerol at least 10 h. The SenSys (Photometrics) and Olympus fluorescent micro- chromosome preparations were observed with BV filter scope (BX51). Pseudocolor images were synthesized by cassette and fluoresce yellow. After the observation, the overlaying the fluorescent staining images and FISH preparations were destained in 45% acetic acid for 30 signals from each probe on the DAPI fluorescence images. min, and ethanol series (30%, 70% and 99.5% (v/v)) for 10 min, respectively. They were treated with McIlvaine’s Chromosome measurements Karyomorphological pro- buffer for 30 min. And then, the slides were stained with perties were based on the data of measurements of the 0.1 µg/ml DAPI in McIlvaine’s buffer for 10 min. After chromosomes at mitotic metaphase taken from the digital the treatment, they were irradiated with U filter cassette photograph images. and fluoresce blue. Photographs were taken with SenSys (Photometrics) and Olympus fluorescent microscope RESULTS AND DISCUSSION (BX51). Chromosome identification followed the proce- The results of somatic chromosomes in two species of C. dure of Hoshi et al. (1999). melo and C. metuliferus are shown in Figs. 1-6, and Table 1. The chromosome number of 2n=24 were counted in PCR amplification Total genomic DNA was extracted both of the two species (Figs. 1 and 4, Table 1). from young leaves, according to the method of Shaw In Cucumis melo L., average chromosome length of (1988). PCR amplification of 5S and 45S rDNA followed the mitotic metaphase was 1.5 µm, and total chromosome the procedure of Hizume (1993) and Sogin (1990), length of the complement was 35.6 µm (Table 1). Size of respectively. The PCR fragments for dual FISH were the longest chromosome was 2.1 µm, while that of the labeled with digoxgenin-11-dUTP (Roche) or biotin-16- shortest chromosome was 1.0 µm (Table 1). The ratio of dUTP (Roche) by means of random primer DNA labeling. the longest chromosome to the shortest chromosome was 2.2 (Table 1). All chromosomes possessed primary FISH procedures For fluorescence in situ hybridization constrictions at the middle parts, the near middle parts or (FISH), the slides were pretreated with 100 µg/ml RNase the near ends of the lengths (Fig. 1). Four satellites were in 2xSSC at 37°C for 1 to 3 h, washed in 2xSSC for 10 seen at the distal regions of short arms on chromosomes + min. Slides were fixed in 4% paraformaldehyde at room (Fig. 1). Chromomycin A3 positive (CMA ) and temperature for 10 min, and denatured at 72°C for 30 sec 4’,6-diamidino-2-phenylindole negative (DAPI−) sites in 70% formamide. Then, the slides were rapidly-cooled were located to centromere and satellite positions (Fig. 1). in ethanol series (70%, 99.5%) at −20°C. Hybridization Two satellites had CMA+ DAPI− nature (Fig. 1, arrows), mixtures contained 50% formamide, 10% dextran sulfate while other two satellites had non-specific nature as CMA and each labeled DNA or fragment at a concentration of 4 or DAPI (Fig. 1, arrowheads). Fluorescence in situ ng/µl in 2xSSC. 25 µl of hybridization mixture was put on hybridization (FISH) signals of two 5S rDNAs and four a chromosomal preparation and covered with a cover slip 45S rDNAs were observed at the ends of chromosomes and sealed with rubber gum. The slides were denatured at (Figs. 2 and 3). The 5S rDNA and the 45S rDNA signals 80°C for 3 min on hot plate and then incubated overnight were located on different chromosomes (Figs. 2 and 3). In at 37°C in humid chamber. After overnight incubation, the 45S rDNA, additionally, two signals were major, coverslips were floated off in 2xSSC, and slides were while other two signals were minor (Figs. 2 and 3). As the washed in 0.2xSSC at 42°C for 15 min twice, 2xSSC at results of FISH, three pares of metaphase chromosomes 42°C for 10 min, and 2xSSC with 0.2% Triton X-100 at were easily identified. Moreover, some characteristic room temperature for 10 min.

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