| In this study, the fluorescence in situ hybridization and in-situ PCR were used to locate the 15 markers from two linkage groups of the Hevea brasiliensis molecular genetic map which were constructed by Lespinasse etc, and these markers of two linkage group were integrated with rubber tree karyotype diagram. The cytological features of molecular biology and chromosome were linked, so it can apply to some structural features characteristic of Hevea brasiliensis chromosome and molecular and cell biology research.It not only lay the foundation for the construction of molecular cytogenetic map of the rubber tree, but also provide the scientific basis for rubber tree molecular assisted breeding,identification of germplasm resources, comparative genomics, comparative genomics. The main results of this study are as follows:First, 14 RFLP markers and 1 SSR maker were selected from two linkage groups of Hevea brasiliensis molecular genetic map by Lespinasse,etc. Among these makers,gHbCIR 637, gHbCIR 240, gHbCIR 486, gHbCIR 208, gHbCIR 495, gHbCIR 657,gHbCIR 461 and gHbCIR 566 are from the linkage groups 5, and others were came from linkage groups 10, they were gHbCIR 113, gHbCIR 347, gHbCIR 168, M 338, gHbCIR 570, gHbCIR 36 and gHbCIR 82. The 30 pairs of primers were designed from these marker sequences,after screening,15 pairs of primers of them were verified very well,that is, a pair of specific primer was screened for each genetic marker. The genomic DNA of Reyan 7-33-97 was used as a template for PCR amplification with each of these 15 pairs of specific primers, subjected to electrophoresis detected after PCR amplification.Electrophoresis of the PCR products was identified, the results of gel electrophoresis was consistent with expectations that the products of 15 pairs of primers were only a clear and single band, and the PCR products and target bands were recovered, sequenced. The sequencing results were compared and the results showed that the sequences amplified by PCR were the target sequences.Second, the makers gHbCIR 495 and gHbCIR 461 from the linkage group 5 were detected by in situ PCR, and the PCR products of the other six markers from linkage groups 5 were purified by gel extraction kit to fluorescently labeled,so it can prepare for probe,next,Fluorescence in situ hybridization experiments. The results of karyotyping showed that the eight genetic markers were located on chromosome 2 of the rubber tree Reyan 7-33-97, and the markers gHbCIR 657, gHbCIR 461,and gHbCIR 566 were located on the long arm of the chromosome 2,the percentage distances from centromere to signal was 30.365, 94.011, 95.625, espectively. While, The markers gHbCIR 64, gHbCIR 24,gHbCIR 49, gHbCIR 208 and gHbCIR 495 were located on the short arm of the chromosome 2, the percentage distances from centromere to signal was 70.33, 51.99,47.16,16.49, 15.53, espectively. It shows that the linkage group 5 of the molecular genetic map which were constructed by Lespinasse et al (2000) was assigned to chromosome 2.Last, the makers gHbCIR 113 and M 338 from the linkage group 10 were detected by in-situ PCR, and the PCR products of the other five markers from linkage groups 10 were purified by gel extraction kit to fluorescently labeled, so it can prepare for probe, next,karyotyping Fluorescence in situ hybridization experiments. The results of karyotyping showed that the seven genetic markers were located on chromosome 4 of the rubber tree Reyan 7-33-97, and the markers gHbCIR 168, M338, gHbCIR 570, gHbCIR 36 and gHbCIR 82 were located on the long arm of the chromosome 4, the percentage distances from centromere to signal was 21.19, 23.35, 25.44, 31.69, 80.60, espectively. While, The markers gHbCIR 631137 and gHbCIR 347 were located on the short arm of the chromosome 4,the percentage distances from centromere to signal was 90.35,69.99,espectively. In other words, the seven markers from linkage group 10 all were located on the chromosome 4 of the rubber tree Reyan 7-33-97, it shows that the linkage group 10 was assigned to chromosome 4. |
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