| Buxus sinic var.parvifolia is one of state-protected trees species. Its distribution regions are narrow, and many of them were dug for potted landscape in the recent years, so the resources are endangered. In order to study the relationship of the clones, and to effectively conserve and utilize the superior genetic resources, fingerprints of clones of B. sinica var.parvifolia were analyzed by techniques of isozyme and ISSR markers,assistanted with morphological markers in this paper.The main results were summarized as follows:1. The peroxidase isozyme patterns in clones of B. sinic var.parvifolia were analyzed by polyacrylamide electrophoresis and only 4 isozyme bands were found. Each clone could be distinguished from the degree of dyeing, but the results were influenced by the factors of environments, which would not exactly differ clones from each other.2. To get high-quality DNA from young leaves of B. sinic var.parvifolia, four different DNA-extracting methods were compared in this paper. The results showed that: The 2% CTAB was the best method to get high-quality DNA. Based on this, the ISSR-PCR reaction system was optimized with primer (CT)8G by the single factor experiment. The system was: 30 ng genomic DNA, 2.0mmol/LMg2+, 0.30μmol/L primer, 0.20mmol/LdNTPs, 1U Taq polymerase with total 20μl reaction solution.3. Nineteen polymorphic primers were screened from 100 primers, there were 256 reproducible and clear amplification products, 211 of which were polymorphic (82.4%), the average number of DNA bands amplified by each primer was 13.5. The DNA profiles based on UBC848 have revealed potential diagnostic fingerprints and specific bands for each clones.4. Assistant with morphological markers, clones of B. sinic var.parvifolia were analyzed by isozyme markers and ISSR markers. It was hard for the isozyme markers to reveal the differences among those with very close relationship since lack of markers sites the single enzyme could afford. With adequate mark sites, the ISSR marker analysis was competent for precise revealing differences and relationships among the clones of B. sinic var.parvifolia. So the ISSR markers might be better in reflecting the relationship of clones. |
