Karyotype Analysis of Wild Rose Species Belonging to Septets B, C, and D by Molecular Cytogenetic Method

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1 Breeding Science 53 : (2003) Note Karyotype Analysis of Wild Rose Species Belonging to Septets B, C, and D by Molecular Cytogenetic Method Maiko Akasaka* 1), Yoshihiro Ueda 2) and Takato Koba 3) 1) Laboratory of Genetics and Plant Breeding, Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo, Chiba , Japan 2) Laboratory of Floricultural Science, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba , Japan 3) Laboratory of Genetics and Plant Breeding, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba , Japan Key Words: Rosa, karyotype, condensation pattern, 45S and 5S rdnas, FISH. The genus Rosa contains more than 150 species that are widely distributed throughout the Northern Hemisphere. Their basic chromosome number is seven, and their ploidy levels range from diploid to octoploid species. Hurst (1925, 1928) considered the basic chromosome number septet as a unit, and he classified diploid rose species into five categories, septets A to E, based on the morphological, physiological and ecological characteristics, mitotic and meiotic chromosome configurations and genetical tests. Polyploid species have more than two identical or different septets. Modern cultivated roses were developed through crosses among only eight diploid species, which are all classified into septet A (Wylie 1954). Chromosomal information is an important key for the classification, phylogenetic analysis and evolutionary biology of plant species, as well as for their morphological and physiological characteristics. Until recently, components of metaphase chromosomes, i.e., number, length and arm ratio, had been commonly used to identify the karyotype of a species, except for maize and rice pachytene chromosomes. Especially in the case of horticultural plants, whose metaphase chromosomes are relatively small compared to those of other plant species, it had been difficult to discriminate individual chromosomes. However, since Fukui and Mukai (1988) developed a method for analyzing prometaphase chromosomes, ideograms of many plant species with small chromosomes have been constructed (for review, see Fukui et al. 2000). In order to clarify the phylogenetic relationships among the wild rose species, we attempted to compare the karyotypes and the physical location of the genes with repetitive sequences. Since the chromosomes of wild roses are small at the mitotic metaphase stage, it was difficult to discriminate Communicated by Y. Furuta Received January 8, Accepted January 30, *Corresponding author ( myco@graduate.chiba-u.jp) each of the chromosomes. However, in our previous studies (Akasaka et al. 2002), ideograms were successfully constructed for five wild rose species by observing the prometaphase chromosomes, and rrna genes were located on the ideograms. In the present paper, the characteristics of the karyotypes of four wild rose species belonging to septets B, C, and D are described. The four wild rose species used in this study are listed in Table 1. All the plant materials were grown from cut seedlings in pots in a greenhouse located at the Faculty of Horticulture, Chiba University, Japan. Root tips were collected from the seedlings showing vigorous growth, treated with distilled water at 4 C for h and fixed in Farmer s fluid (ethanol : acetic acid = 3 : 1) at 4 C for at least 2 days. After treatment with an enzyme mixture consisting of 3 %(w/v) Cellulase Onozuka RS, Yakult, 1.5 %(w/v) Pectolyase Y-23, Seishin, in 0.01 M sodium citrate, ph 4.6 at 37 C for min, the root tips were placed on glass slides and exposed to steam over hot water at 76 C for 2 sec. Then, they were tapped with a forceps in a few drops of the fixative (methanol : acetic acid = 3 : 1), re-exposed to steam and air-dried on a hot plate at 60 C for 2 min. Chromosomes were stained with a 1 %(v/v) Giemsa (Merck) solution in 1/15 M sodium phosphate buffer (ph 6.8) for two min. Six well spread prometaphase plates were selected under a microscope (BH2, Olympus) and photographed on black and white film (Minicopy F, ISO 64, Fuji Film). Analysis of the condensation patterns of Giemsastained chromosomes was performed using Chromosome Image Analyzing System III (CHIAS III, Kato and Fukui 1998) with a public domain program, NIH image. Homologous chromosomes were detected by observing the morphological characteristics and condensation patterns, and designated as Chromosomes 1 to 7 according to their total length, excluding the satellite regions. For FISH analyses, the 45S rdna clone from wheat (pwrrn) which was originally isolated by Barker et al. (1988) (kindly provided by Dr. H. Tsujimoto, Tottori Univ.) and the 5S rdna clone from R. multiflora were used as probes. The 45S rdna was labeled with biotin-16-dutp (Roche) by the nick translation method, while the 5S rdna was amplified and labeled with digoxigenin-11-dutp

2 178 Akasaka, Ueda and Koba Table 1. Rosa species used in the karyotype analysis Species Septet Chromosome number Distribution Chiba Univ. accession number Genus Rosa Subgenus Rosa Section Rosa R. willmottiae B 2n = 14 Western Chiba KRN 1), Chiba R. rugosa C 2n = 14 Aomori, Japan 85P-85 R. marretii D 2n = 14 Nagano, Japan 96UN-NA-16 Section Carolinae R. foliolosa D 2n = 14 Southeastern North America KRN 1), Chiba 1) KRN = Keisei Rose Nursery (Roche) by the PCR labeling method. FISH was performed following the protocol of Ohmido and Fukui (1997). Chromosomes were counterstained with 4,6 diamidino-2- phenylindole (DAPI). Each fluorescent signal was independently observed using a fluorescence microscope (BH2-RFCA, Olympus). The image of each fluorescent signal was photographed separately on a color film (SPERIA F, ISO400, Fuji Film), recorded and processed using Adobe PhotoShop 5.0 software (Adobe Systems Inc.). Fig. 1 shows the images of prometaphase chromosomes of the four wild rose species, in which seven chromosome pairs could be discriminated from each other. In all the species, satellite chromosomes designated as Chromosome 7 were the shortest among the 14 chromosomes. Table 2 lists the relative length of the rose chromosomes and chromosome arms. The relative length of the chromosomes varied continuously from 10 to 20 % in the four species. Arm ratios are shown to determine the position of the centromeres. The largest two chromosomes in each species were metacentric or median, and the others were submetacentric or subterminal. Fig. 1. Giemsa-stained prometaphase chromosomes of A: Rosa willmottiae, B: R. rugosa, C: R. marretii and D: R. foliolosa. Numerals indicate the chromosome numbers used in the present study. Bar = 5 µm.

3 Karyotype analysis of wild roses 179 There were no acrocentric or telocentric chromosomes in the four rose species studied. Fig. 2 shows the physical location of the 45S and 5S RNA genes on the chromosomes. A pair of 45S rdna sites was observed in R. willmottiae, R. rugosa and R. marretii, while three pairs of 45S rdna sites were found in R. foliolosa. A pair of 5S rdna sites was observed on the chromosomes with medium length in all the species. Fig. 3 shows the ideograms of the chromosomes constructed based on the density profiles, which reflect the condensation patterns (CP) of the chromatids and the physical location of the rrna genes. In all the species studied, chromosome 7 showed a heterogeneity in the morphological characteristics. Characteristics of the chromosomes of each species were as follows: i) R. willmottiae Except for one counterpart of Chromosome 7, all the chromosomes showed heavily condensed proximal regions including the centromeric regions. Chromosomes 1 and 2 were median and showed similar condensation patterns. Chromosome 3 was subterminal and its long arm was the longest among the chromosomes, resulting in the smallest arm ratio. Chromosomes 4, 5 and 6 were submedian and showed similar condensation patterns. One pair of 5S rdna sites was detected in the proximal region of the long arm of Chromosome 4. The Chromosome 7 pair was heteromorphic, and the sizes of the satellite were also different. Two 45S rdna signals were observed at the end of the short arms. ii) R. rugosa All the chromosomes showed heavily condensed proximal regions. Chromosome 1 and Chromosome 2 were median and submedian chromosomes, respectively. Chromosomes 3, 4, 5 and 6 showed similar condensation patterns but differences in the arm ratio and the length of the condensed regions. Two 5S rdna sites were detected in the proximal region of the long arm of Chromosome 5. The Chromosome 7 pair was heteromorphic, showing differences in the total length and satellite size between the homologues and a heavily stained spot in only one of the satellites. Two large 45S rdna sites were observed at the end of the short arms. iii) R. marretii In all the chromosomes, large portions of the proximal region of the short arms were condensed, while only small portions were heavily stained on the long arms. Chromosomes 1 and 2 were median chromosomes whose proximal region was highly condensed. Chromosomes 3, 4, 5 and 6 were submedian pairs. Two 5S rdna sites were detected in the proximal region of the long arm of Chromosome 4. In Chromosome 7, the total length, size of the satellites and location of the 45S rdna regions were different between the homologues. iv) R. foliolosa All the chromosome arms were nearly occupied with condensed chromatin except for the terminal regions. Chromosomes 1 and 2 were median chromosomes, with highly condensed proximal regions. Chromosome 3 was a sub- Fig. 2. Fluorescence in situ hybridization of rose chromosomes using 45S and 5S rdnas as probes. A: Rosa. willmottiae, B: R. rugosa, C: R. marretii and D: R. foliolosa. These figures show FITC- (yellow) and Texas red- (red) stained signals of 45S and 5S rdnas labeled with biotin and digoxigenin, respectively. Bars = 5 µm.

4 180 Akasaka, Ueda and Koba Fig. 3. Ideograms of chromosomes of A: Rosa willmottiae, B: R. rugosa, C: R. marretii and D: R. foliolosa, constructed based on density profiles. Numerals denote the chromosome numbers used according to their relative length in a cell. Black, gray and white areas represent chromatin regions with heavy, intermediate and slight condensation, respectively. The gaps in the middle of the chromosomes indicate the centromeric regions. Double and single circles denote the position of the 45S and 5S rrna loci, respectively. Individual homologues are indicated for heterologous pairs. median chromosome with a 45S rdna site observed at the end of the short arm. No clear constriction was observed in this region. Chromosome 4 was submedian and had a 45S rdna site at the end of the short arm, showing a second constriction in this region. A pair of 5S rdna signals was detected on the long arm of Chromosome 4. Chromosomes 5 and 6 were submedian pairs with large heterochromatic regions on the short arms. Chromosome 7 consisted of a submedian pair with differences in the total length of the chromosomes between the homologues. 45S rdna sites were observed at the end of the short arms that showed second constrictions. Ma et al. (1997) and Fernandes-Romero et al. (2001) located the sites of the 45S rrna genes by FISH in wild and cultivated rose species. Their results revealed the existence of a pair of 45S rdna signals in the terminal region of a pair of chromosomes, but, they could not discriminate the chromosomes. In the present study, we confirmed and located the two signals on the shortest chromosomes with satellites in R. willmottiae, R. rugosa and R. marretii. However, six 45S rdna sites were observed in R. foliolosa, which is endemic to eastern North America. Flory (1950) reported the geographical distribution of diploid and polyploid rose species and concluded that Rosa originated in East Asia and that some forms migrated to North America. The present results suggest that some chromosomal rearrangements may have occurred in R. foliolosa during its migration to North America, and that karyotype differences may be found even within the same septet, although Wu et al. (2001) reported that the sections Rosa and Carolinae were closely related to each other, based on the sequences of the matk gene. The wild rose species in the section Carolinae including R. foliolosa are distributed only in North America and R. foliolosa was

5 Karyotype analysis of wild roses 181 Table 2. Relative length of the rose chromosomes and chromosome arms (Total length in a cell = 100) Species Chromosome No. Short arm 1) Long arm 1) Total Arm ratio 2) R. willmottiae Total R. rugosa Total R. marretii Total R. foliolosa Total ) The data represent the averages of twenty four chromatids per one chromosome on six prometaphase plates. The length of the satellite was excluded from the calculation. 2) Short arm length/total length used in the present study for karyotype analysis for the first time. The other species in the section Carolinae should also be investigated. Recently, geographical differences in karyotypes have been recorded in Trillium and Avena. Fukuda (2001a, 2001b) reported in two autopolyploid Trillium species that chromosome rearrangement had occurred in either or both T. undulatum in America and T. govanianum in Asia. In Avena agadiriana, Hayasaki et al. (2001) reported differences in the FISH signals of the 45S rdna loci among five accessions of tetraploid species distributed in different regions of Morocco. The present study has provided information about the karyotype analysis of wild rose species by using prometaphase chromosomes and has shown that chromosome analysis could be performed, even in species with small chromosomes. In addition to the quantitative analysis of the condensation pattern, more information about the physical location of genes should be obtained to reveal chromosome rearrangements and to perform a precise karyotype analysis of rose species. Acknowledgements We thank Dr. M. Ito, Nara Institute of Science and Technology, and Dr. M. Mishima, Kyushu University, for their valuable suggestions. This work was supported (in part) by Sasakawa Scientific Research Grant from Japan Science Society to M.A. Literature Cited Akasaka, M., Y. Ueda and T. Koba (2002) Karyotype analyses of five wild rose species belonging to septet A by fluorescence in situ hybridization. Chromosome Science 6: Barker, R.F., N.P. Harbend, M.G. Jarvis and R.B. Flavell (1988) Structure and evolution of the intergenic region in a ribosomal DNA

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