| In this study, physical mapping of six BAC clones containing rice resistance genes was comparatively carried out between Oryza sativa L. subsp. indica Kato var. Guangluai No. 4 and Oryza granulata Nees et Arn. ex Watt. by fluorescence in situ hybridization (FISH) technique. The FISH signal features of centromere, tclomere and rDNAs repeated sequences were charactered in O. granulata. Genomic components of interspecific hybrid F1 of O. sativa and O. granulata were also identified by genomic in situ hybridization (G1SH). The main results are as follows:Used Southern blotting analysis- the genomic homologies of the RFLP makers linked to resistance genes Gm-2, Gm-6, Pi-5(t), Bph3, xa-5, Glh and RTSV were tested between O. sativa and O. granulata. Clear hybridization bands of the tested RFLP makers were observed for all treatments of DNA samples from the two species. These results suggested that sequences homologous to the tested RFLP markers existed in O. granulata.The rice BAC clones 31E20, 24E21, 4F22, 38J9, 14E16, and 44B4. which contain rice resistance genes and centromere clone pRCS2 were comparatively mapped between O. sativa and O. granulata. Clear hybridization signals were detected for all the BAC clones in interphase nuclei, mitotic chromosomes and meiotic pachytene chromosomes of these two species.The BAC clones 31E20, 24E21, 4F22, 38J9 and 14E16 were located on chromosome 4 of 0. sativa. The FL (fraction length, percentage of the distance from FISH hybridization site to the end of the short arm of the chromosome divided by the total length of the detected chromosome) value of detected signals was 65.2 ± 2.72%, 59.8±2.13%, 46.4 ± 1.72%, 16.8±1.13% and 5.7 ± 0.72% correspondingly, FL value of the centromere on the chromosome 4 of O. sativa was 17.8 ± 2.45%.The six rice BAC clone probes were also hybridized respectively onto pachytene chromosomes of O. granulata and the FL value of 31E20, 24E21, 4F22, 38J9 and 14E16 were 64.1 ± 2.45%, 58.5 + 1.66%, 44.5 ± 2.31%, 17.5 ± 1.36% and 5.2 ± 0.95%, respectively. Dual FISH signals showed that the FL value of the centromere of the chromosomes showing 31E20 signals was 17.4+1.76%. BAC clone 44B4 was located on the terminal of short arm chromosome 5 in O. sativa and on that of short arm of a given chromosome in O. granulata.The morphologies of the chromosomes located by 5 of the tested BAC clones in O. granulata were very similar to those of chromosome 4 of O. sativa containing these clones. To determine whether these clones were located on the same chromosome in O. granulata, dual color FISH were performed on pachytene chromosomes. The results indicated that all the tested clones were located on the same chromosome of O. granulata. The FL value for hybridization signals of the tested clones in dual color FISH was basically consistent with that observed in separate hybridization. oThe Southern analysis and FISH results demonstrated that high sequences homology, synteny and colinearity for the tested clones existed between O. sativa and O. granulata. Moreover, the relative positions of the tested five BAC clones were basically similar between O. granulata and O. sativa. These two species contain different genomes, AA and GG, respectively and have evident differences of botanical features and ecological habits, but they were conservative in sequences and distribution of the tested clones on the chromosomes.RCS2 is a tandem repetitive DNA sequence and it is the marker of the centromeres in O. sativa. FISH results of the centromere sequence pRCS2 on mitotic chromosomes of O. granulata showed that the hybridization signals were located at the centromeric regions of all the chromosomes. In addition, no signal was shown elsewhere and each chromosome showed only one signal site. The different chromosomes showed prominent differences in the strength of the signals, so did those in O. sativa. Therefore, the sequences of RCS2 were conserved between those two species.FISH of the telomere sequences showed that the signals displayed on the ends of different chromosomal arms and they were not the same in the size and strength, and heteromorphisms existed on two members of the homologue. Some of the signals displayed two or three segments on the chromosome ends of the root tip cells. However, the multiplesegment signals disappeared and the signals diminished and weakened in the pollen mother cells.In O. gramdata, 5S rDNA showed one signal site and it was detected on the region close to the centromere of the short arm on a given mitosis and pachytene chromosomes. FL value of 5S rDNA on pachytene chromosomes was 38.7 + 4.2 %. Both the mitotic and pachytene chromosomes carrying 5S rDNA signals resembled the chromosome 11 of O. saliva containing 5S rDNA according to their relative length and arm ratio. Moreover O. gramdata was similar to O. sativa in the relative position of 5S rDNA on the chromosomes.One signal site of 45 S rDNA was detected at the terminal region of the short arms of a given mitotic chromosome pair in O. gramdata. The detected chromosome was similar to chromosome 9 of O. sativa containing 45S rDNA in its related length and arm ratio, which were 6.67 ± 0.47 % and 4.42 ± 1.72.The intensity of 45S rDNA signals displayed striking differences between two members of the detected homologue. One of the two members showed a very large and intensive block signal, and the other showed very small and faint one. FISH on pachytene chromosomes further verified the above results and the two homologues carrying 45S rDNA paired incompletely. The large signal was equal to about 33.3± 9.9 % of the total chromosome length and the small signals on the other homologue was about 2-3%.In O. gramdata fiber FISH of 45S rDNA indicated the signals basically included two classes. One showed long signals, which were roughly, estimated its size at over 1Mb. The other showed short string signals and at 84.9 ± 10.3 kb. Based on the data of the Southern analysis, copy number of the 45S rDNA fiber was estimated. Used the techniques combining FISH and Ag-staining the relationship between the copy number of 45S rDNA and nucleolus formation was illustrated in a certain degree.GISH using Bio-labeled DNA of O. gramdata DNA with no blocking DNA of O. sativa demonstrated that there were more prominent differences between genomes A and G. In the F] hybrid, dual FISH showed that the chromosomes of O. gramdata were larger than those of O. sativa in length and heterochromatin contents. The chromosomes with corresponding order, which was arranged based on the length, were compared between O. gramdata and O. sativa and their chromosomal lengths were different obviously. The chromosome 4 of O. gramdata is 1.98 times longer than that of O. sativa in relative length.The synapsis between genomes A and G could be observed more effectively with dual FISH. During pachytene stage most of the chromosomes between A and G could not pair each other. The synapsis in distal regions and the partial bivalents derived from the synapsis were observed only for one or two chromosomes. There were synapsis phenomena between its own chromosomes in each of the two species. It demonstrated that some homology existed between nonhomologous chromosomes in both A and G.In this study there were following innovation results. Firstly, comparative cytogenetic investigations of mitotic and meiotic chromosomes in O. granulata, O. sativa and their hybrid Fi were reported for the first time. Secondly, the relationship between heteromorphism of cope number of 45S rDNA and its function was analyzed by the techniques combining FISH with Ag-stain. These results which have never been reported before is quite important for promoting the development of plant molecular cytogenetics. |
PDF Full Text Request