菊池 真司

To investigate the nature of plant chromosomes irradiated by heavy-ion beams, the effects of nitrogen (N) and neon (Ne) ion beams on hexaploid wheat chromosomes were compared with those of X-ray. Chromosome aberrations, such as short, ring and dicentric chromosomes appeared in high frequency. The average numbers of chromosome breaks at LD-50 by irradiation with X-ray, N and Ne ion beams were 32, 20 and 20, respectively. These values may be underestimated because chromosome rearrangement without change in chromosome morphology was not counted. Thus, we subsequently used a wheat line with a pair of extra chromosomes from an alien species (Leymus racemosus) and observed the fate of the irradiated marker chromosomes by genomic in situ hybridization. This analysis revealed that 50 Gy of neon beam induced about eight times more breaks than those induced by X-ray. This result suggests that heavy-ion beams induce chromosome rearrangement in high frequency rather than loss of gene function. This suggests further that most of the novel mutations produced by ion beam irradiation, which have been used in plant breeding, may not be caused by ordinary gene disruption but by chromosome rearrangements. © 2009 Elsevier B.V. All rights reserved.

To investigate the nature of plant chromosomes irradiated by heavy-ion beams, the effects of nitrogen (N) and neon (Ne) ion beams on hexaploid wheat chromosomes were compared with those of X-ray Chromosome aberrations, such as short, ring and dicentric chromosomes appeared in high frequency The average numbers of chromosome breaks at LD-50 by irradiation with X-ray, N and Ne ion beams were 32, 20 and 20, respectively These values may be underestimated because chromosome rearrangement without change in chromosome morphology was not counted. Thus, we subsequently used a wheat line with a pair of extra chromosomes from an alien species (Leymus racemosus) and observed the fate of the irradiated marker chromosomes by genomic in situ hybridization This analysis revealed that 50 Gy of neon beam induced about eight times more breaks than those induced by X-ray. This result suggests that heavy-ion beams induce chromosome rearrangement in high frequency rather than loss of gene function. This suggests further that most of the novel mutations produced by ion beam irradiation, which have been used in plant breeding, may not be caused by ordinary gene disruption but by chromosome rearrangements. (C) 2009 Elsevier B V All rights reserved.

We determined the numbers and locations of 5S and 45S ribosomal RNA genes (rDNAs) in seven Fagopyrum species, three major and four wild buckwheat species, using fluorescent in situ hybridization (FISH). Although the chromosome numbers between diploid Fagopyrum species were conserved, there was variation in both number and location of rDNA loci. Common buckwheat, F. esculentum, contained three 5S and three 45S rDNA loci allowing us to identify five out of eight pairs of the chromosomes by the rDNA pattern. In addition, F. tataricum and F. cymosum carried one 5S and two 45S rDNA loci, which allowed us to identify three out of eight pairs of the chromosomes in the two species. The rDNA loci of four wild buckwheat species, F. urophyllum, F. pleioramosum, F. callianthum and F. leptopodum showed diversified location. The distribution of the rDNA loci implies a chromosome evolution in Fagopyrum via larger structural rearrangements such as inversions and translocations. The numbers and the locations of rDNAs support the phylogenetic relationships of buckwheat based on the data of internal transcribed spacer (ITS) region of rDNA.

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and. T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.

The precise guidance of pollen tubes is important for successful fertilization. Based on abnormalities in interspecific and intergeneric crosses, we investigated pollen tube guidance in Torenia fournieri, which is a model plant for studies on plant fertilization due to its protruding embryo sac. Critical abnornmalities of pollen tube growth have never been recorded in interspecific crosses between either T baillonii or T concolor, and hybrids could be obtained. In contrast, abnormalities appeared in intergeneric crosses with twelve other species. The abnormalities could be observed for five phases of pollen tube guidance revealed in Arabidopsis mutants as follows: germination of the pollen grains, penetration of pollen tube through stigma cells, growth in the transmitting tract, arrival at the placenta and release of sperm into the micropyle. In the self-pollination of T fournieri, in vivo, the pollen tubes grew straight from the placenta to the protruding embryo sac, omitting funiculus guidance. In addition, observation of an intergeneric cross between T fournieri (female) and Mimulus hybridas revealed that 64% of the pollen tubes grew straight to the micropyles of ovules rather than to synergid cells, suggesting that the micropyle in T. fournieri transmits the attraction signal. In addition, the signal attracted pollen tubes of M. hybridas to the inicropyle, unlike those of T. fournieri.

In this study, we investigated chromosome variation of ten Pennisetum species by fluorescence in situ hybridization (FISH) probed with a highly repetitive centromeric satellite and 45S rDNA sequences. We also analyzed genetic relationship using EST microsatellite markers derived from pearl millet. FISH with the centromeric satellite sequence showed strong to medium hybridization signals at the centromeric regions of all the chromosomes in six species, weak signals on two chromosome pairs in one species and no signals in three species. Locations of 45S rDNA varied both in number and strength among the species. Two species showed signals on one chromosome pair, five species on two chromosome pairs, one species on three chromosome pairs and two species on four chromosome pairs. In addition, DAPI staining exhibited heterochromatin blocks at centromeric, telomeric or both regions in the tested species. Prominent signals were observed at telomeric regions in two species. Polymorphism among the EST-microsatellite markers and phylogenetic tree of these species fitted well with the chromosome variation of these species. The discrepancy between the morphological classification and the phylogenetical grouping suggests that the classification of Pennisetum species should also be considered at the genomic level as well as the morphological features.

Torenia is a suitable model plant to study plant fertilization because of its protruding embryo sac. However, information on the genomes and chromosomes of this species is limited. We determined the genome sizes of T. fournieri Linden and T. baillonii Godefr as 1.71 pg x 10(8) bp and 1.67 x 10(8) bp, respectively. The small genome size of these species suggests their superiority as the targets for molecular cloning studies. Furthermore, karyotypes of T. fournieri and T. baillonii were determined using FISH probed with 5S rDNA, 45S rDNA and species-specific centromere repetitive sequences. Although the two species have similar genome size, number of chromosomes, centromere repeats and 5S rDNA loci were varied. Observation of meiosis in the F-1 hybrid revealed that all chromosomes except one of T. fournieri paired well with the chromosomes of T. baillonii throughout the entire length of the chromosomes including species-specific centromeric regions. One exceptional chromosome of T. fournieri behaved as a univalent and was not always required for gametogenesis. The present results provide the basis for the molecular genetics in Torenia.

Torenia fournieri is a good model plant to study fertilization in plants because it is easy to observe the fertilization process due to the protruding nature of the embryo sac. To study the location and movement of chromosomes and their centromeres in early embryogenesis of interspecific hybrid plants, we isolated two families of centromere-specific tandem repetitive sequences from T. fournieri and T. bailonii, and named them the "TCEN-family" and "BCEN-family", respectively. Both sequences consisted of a repeat unit of 52 bp located in the pericentric and centric heterochromatins. All signals of both sequences were prominent, but their intensity varied among the chromosomes. DNA-blot hybridization indicated the presence of similar sequences of TCEN-family in T. concolor, N. caerulea, and "Summer Wave", whereas the BCEN-family was found only in T. bailonii, thus indicating the wide or specific distribution of their repetitive families observed. We also applied whole-mount FISH to the interspecific hybrid embryos by using TCEN- and BCEN-family sequences as probes. Our results suggest that whole-mount FISH with the species-specific centromere sequences as probes is an ideal method to analyze the dynamics of chromosomes and centromeres in interspecific fertilization and early embryogenesis. Copyright (C) 2005 S. Karger AG, Basel.