Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat ( PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.

Summary Every species has a unique karyotype, but certain genera have common karyptypes among species. The markers (chromosome length and centromere position) used in traditional karyotyping do not distinguish all chromosome pairs in the genus Pinus. However, the application of multi-probe fluorescence in situ hybridisation (FISH) procedures allowed exact karyotyping of 26 Pinus congeners. We used these new data to examine species relationships. The 35S rDNA and 5S rDNA, Arabidopsis-type telomere repeat sequences, and the proximal CMA band-specific repeat (PCSR) of P. densiflora were used as FISH probes for our analysis of chromosomes in 26 Pinus species. Each species had a unique FISH karyotype and most homologous chromosome pairs were identified. The FISH karyotypes were used to compare corresponding or homologous chromosomes among the species. Common or similar FISH signal patterns appeared in closely related species. Species that had inherited common FISH signal patterns were classified into four karyotype groups. We used cluster analyses to compare quantitative differences in FISH signals within these groups. The results of these analyses were consistent with recent systematic interpretations and resolved differences among existing taxonomic systems based on diverse methodologies. Our results indicate that FISH signal patterns reflect the history of species differentiation and that comparative FISH karyotyping has potential as an important tool for studying the taxonomy or phylogeny of Pinus.

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8-76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.

Summary Nigella damascena has 2n=12 chromosomes composed of five long metacentric chromosome pairs and a short telocentric chromosome pair. FISH using rDNA probes was applied on the chromosomes of this species. 45S rDNA were located at the terminal region of three pairs of long metacentric chromosomes and a pair of short chromosomes. 5S rDNA appeared at the interstitial regions of the short arm of two long metacentric chromosomes. Chromosome, FISH, Nigella damascena, 45S and 5S rDNA. Nigella damascena is one of the 14 species that belong to the genus Nigella. It is planted in gardens all over Japan as an ornamental plant. The Nigella species are diploid and its genome (2n=12) has five pairs of long metacentric chromosomes and a short telocentric pair. Therefore, these species were used preferentially in early studies for the early stages of comparative karyotype analysis to study plant chromosome structures and the effects of mutagens or irradiation on chromosomes (Gregory 1941, Kurita 1956, 1959, Moutschen-Dahmen and Moutschen-Dahmen 1965, Moutschen 1968, Moutschen et al. 1969, Marks 1975. Previous studies showed the karyotype, localization of heterochromatin or C-bands (Marks 1975), NORs by silver staining (Hizume et al. 1982), and fluorescent bands by base-specific fluorochromes (Hizume et al. 1989). Modern methods have not been applied to the chromosomes of this species at all. The location of rRNA genes has not been reported until now. We report the locations of 45S rDNA and 5S rDNA on the chromosomes by using FISH, and compare them with previous chromosome studies. Key words Materials and methodsSeeds of Nigella damascena cv. Persian Jewel were obtained commercially from Sakata Seed Company. The seeds were germinated on wet filter paper in the dark at 20 o C. The primary root tips were collected and treated in 0.05% colchicine for 3 h then fixed in acetic alcohol (1 : 3). Chromosomes were prepared by enzymatic maceration in 1% Pectolyase Y-23 and 4% Cellulase R10 at 37 o C for 30-40 min, followed by air or flame drying. rDNAs were amplified by the CTAB method using genomic DNA extracted from young leaves of the species as the template. DNAs were labeled with biotin or digoxigenin and used as probes for two-color FISH. The PCR, labeling, and FISH procedures are conducted according to a previous report (Hizume et al. 2002). Chromosome images and FISH signal features were captured by a cooled CCD camera, and the images were processed using IPLab software.

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