Cloning Of Telomere Associated Sequence And Establishment Of Chromosome Specific FISH Markers In Chinese Cabbage

Repeat sequence is one of the important components in eukaryotic genomes. It is not only important to maintain chromosome structure in space, the expression regulation of genes and genetic recombinations, but also tandem repeat DNA cluster often has a specificity of genus, species or even the chromosome. Thus the cloning and analysis of repeat sequence are effective means to research eukaryotic genome. Chinese cabbage is one of the most important vegetable crops in cruciferous Brassica diploid species. In addition, the chromosome of Chinese cabbage is small, and the form nonhomologous chromosome is similar. Establishment of an effective method of chromosome recognition is important for karyotype analysis and cytogenetics research. Up to now, only 6 different chromosomes can be accurately distinguished from the total 10 different chromosomes based on FISH using rDNA as probes. Based on these, using the primer of Arabidopsis telomeric repeat sequence, the genome DNA of Chinese cabbage inbred line‘85-1’was amplified and the telomere repeat sequence was cloned and analyzed. Using the BAC clones anchored different chromosome in Chinese cabbage as probes, the FISH on the Chinese cabbage meteaphase chromosomes was conducted.According to above FISH results, the karyotype distinguished different chromosomes of Chinese cabbage was constructed. The main results are showed as follows:1. Using Arabidopsis telomeric repeat sequence (TTTAGGG)3 as a primer, a DNA fragment with length of 344bp was amplified and cloned from Chinese cabbage. Sequence analysis showed that it is a telomeric repeat sequence (TAS), which is mapped to the proximal end of chromosome 2 of Chinese cabbage. In this TAS fragment, there is Arabidopsis telomeric repeat Sequence (TTTAGGG)3, it also contains telomeric repeat sequence of other creatures, such as silkworm.2. Ten FISH signals of 5S rDNA probe were observed in Chinese cabbage, which were localized the long arms nearly centromere of chromosome 1, 2, 3, 5 and the end short arms of chromosome10. Ten FISH signals of 45S rDNA probe were also observed in Chinese cabbage, which were located on the long arms nearly the centromere of chromosome1~5.3. Sixteen FISH signals of CentBr1 rDNA probe were observed in Chinese cabbage chromosomes, which were localized on the centromere of chromosome1,3,4, 6~10, 4 FISH signals of CentBr2 rDNA probe were observed in Chinese cabbage 10 chromosomes, which were localized on the long arms nearly centromere of chromosome 2 and the end of long arms of chromosome 5.4. Except A1 linkage group, the BAC probes anchored the other nine linkage groups (A2 to A10) of Chinese cabbage displayed a pair of FISH signals in its corresponding chromosome. There into A2~A10 linkage groups were corresponding to chromosome 6, 2, 9, 5, 4, 7, 8, 1 and 10, respectively. A karyotype diagram based on FISH marker of rDNA, CentBr1, CentBr2 and chromosome specific BAC probes was established, which providing a reliablebasis basis for accurate identification of Chinese cabbage chromosomes.