Application Studies Of Fluorescence In Situ Hybridization Technique In Genes Localization On The Chromosomes Of Chinese Cabbage (Brassica Campestris L.ssp.pekinensis Olsson, Syn.B.rapa L.ssp.Pekinensis)

Chinese cabbage is not only an important vegetable crop origined from China but also one of the most important researchful objects of Multinational Brassica Genome Project. Gene localization is the basis for identifying, cloning and utilizing Chinese cabbage genes. In the field of molecular cytogenetics, gene physical orientation with noneuploid and fluorescence in situ hybridization technique is increasingly becoming the hotspot of gene localization research, which is very important to the chromosomal physical mapping, associating the genetic linkage groups with the specific chromosomes, and combining genetic map with physical map. With the diploid and a set of primary trisomics self-established as materials, in this research, cytological identification of genetic stability of the diploid and different trisomics via asexual multi-generation was made with chromosomal karyotype analysis, two rDNA repeat sequence and several functional gene fragments obtained by PCR and cloning were physically localized the specific chromosomes of Chinese cabbage with FISH technique. The study results were as follows:1. With the study of correlative parameters during FISH, the technique system on Chinese cabbage was found, including chromosome preparation, chromosome pretreatment, probe hybridization, washing and signal surveying. Utilizing the new system, 10 signals, which had different fluorescence intensity, was observed successfully on metaphase and interphase chromosomes of Chinese cabbage respectively, which help to the utilization of FISH on Chinese cabbage. The method of obtaining nuclei during fiber-FISH was improved by chopping the fresh young cotyledon with a sharp sterile scalpel, and the smooth DNA fibers were dragged and extended with a clean coverslip. The hybridization signals were inconsecutive long chain like prayer beads with Chinese cabbage genome DNA as a prober.2. The karyotype formula of Chinese cabbage chromosomes 2n=2x=20=10m +8sm+2st(SAT), and the karyotype type is 2B. Chromosome 1, 2, 3, 4, 5 and 7 are all central centromere chromosomes, the centromere of chromosome 3, 6, 8 and 9 lie the approximate central centromere chromosomes, and the centromere of chromosome10 lies near the chromosome end. In general, the bright region stained by DAPI contains richer heterochromatin, which mostly concentrate on the twain side near the centromere. The number and the position of DAPI band are basically consistent between the two homologous chromosomes.3. The number variation of chromosome was observed in some cells of Chinese cabbage root conserved multi-generation recur to tissue culture. But the karyotype analysis of different trisomics indicated that chromosome replacement did not take place among 10 trisomics which chromosome number did not change.4. The gene 25S rDNA from Arabidopsis thanalisis was studied on metaphase chromosome of Chinese cabbage using FISH. The result indicated that there were five homologous region of 25S rDNA in Chinese cabbage genome, which were located on five pairs of chromosomes comprising of chromosome 1, 2, 3, 4 and 10 respectively, and the fluorescence intensity of hybridization signals was various on different chromosomes. The number of 25S rDNA copies in Chinese cabbage genome was confirmed as 566+137 with DNA fiber-FISH technique. But the number of 25S rDNA hybridization signals on different Chinese cabbage trisomics ranged from 7 to 12, located on 4-7 homologous section. In general, 25S rDNA homologous region existed on both the long arms of chromosome 1-4 and the satellite region of chromosome 10 in different trisomics, but the observed frequency of the FISH signals was different on the same chromosome of different trisomics, and the intensity of fluorescence signal was also different, the strongest signal was detected on the satellite region of chromosome 10 in different trisomics (not including triplo4 and triplo5).5. 6 FISH signals of 5S rDNA were observed in the diploid of Chinese cabbage, which were localized on the long arm of chromosome 2, the short arms of chromosome 9 and chromosome 10 respectively. 7, 6 and 7 FISH signals were also observed in triplo2, triplo3 and triplo9 respectively. Furthermore, the chromosomes and the position that these signals were localized on were the same as on the diploid. The number of 5S rDNA copies in Chinese cabbage genome was confirmed as 679 with DNA fiber-FISH technique.6. FISH on metaphase complements, using the 25S and 5S rDNA, produced good diagnostic markers for identifying chromosome 1-4, 9 and 10.7. 14 functional gene fragments were obtained using PCR and gene cloning, which were named EPSP1409- RGA94^ SLG773> BccLH9o4> RPS267i> SALM742^ VDAC994> CYP95^ BcNCED1453^ BcGR803> ME725^ SILi445> B\T>m, FLC909 respectively according to their lengths. The BLAST result of the nucleotide acid sequence showed that these obtained gene fragments (except BcGRscb) were highly homologous with the referenced genes, and the degree ranged from 89% to 99%.One pair of signals of each probe was observed on the metaphase of Chinese cabbage diploid. These signals of four probes containing of EPSPi4os>> RGAg44^ RPS2671 and CYP954 were localized the short arm central section of chromosome 3, the long arm central section of chromosome 3, the long arm terminal section of chromosome 2 and the long arm central section of chromosome 1 respectively. The frequency observed of four probes was all lower and 6.48% averagely. But it was the first of FISH localization on the chromosomes of Chinese cabbage using low copies gene as the prober, which would establish the basis for the localization of functional gene with sigle copy and low copies in Chinese cabbage and the studies comparing with other species.