| As one of the important fruit trees, persimmon was originated in China. It has a large number of cultivars, very long cultivated history and very wide distribution. Because of lacking of standardized naming system, both synonym and homonym are presented very seriously. In addition, the genetic relationships among cultivars were not very clear in persimmon. As a new DNA marker, AFLP (amplified fragment length polymorphism) has been widely used in cultivar identification, genetic diversity, and genetic linkage mapping in fruit trees. In order to provide scientific basis for the identification, collection, and conservation and utilization of persimmon germplasm, AFLP silver-staining technique system suitable for persimmon was established. The fingerprints of 109 persimmon cultivars were constructed, and the genetic diversity of these cultivars was analyzed. The core collection of assayed cultivars was also established based on the AFLP marker and morphological data.1. The genomic DNA of persimmon was isolated using a modified CTAB method to remove its abundant tannin, polysaccharide and pigment. The modified extraction buffer contained 100 mmol/L Tris-HCl (pH 8.0), 20 mmol/L EDTA, 1.4 mol/L NaCl, 2% CTAB, 1% PVP-40, 10 mmol/L Na2S2O5, 100 mmol/L 3 -mercaptoethanol (added before isolation). The RNA was removed from the solution by adding RNase, and the DNA was purified by using phenol/chloroform, chloroform/isoamyl alcohol (twice). The purified genomic DNA was suitable for AFLP analysis in persimmon.2. After systematically study on the main factors involved, an AFLP silver-staining technique system fitting for persimmon genomic DNA analysis was established. Restriction digestion of genomic DNA was performed using two restriction enzymes. Around 450ng DNA was digested with 3 units of EcoR I and 3 units of Mse I enzymes in a reaction volume of 20 uL at 37℃ for 4h. Double-stranded adaptors were legated to the restriction fragments at 37℃ more than 10h (or overnight), then transferred to 65℃ for 10min, after that the digested-legated DNA fragment was diluted. (5 folds) with TE buffer and used as templates for pre-amplification. The pre-amplification was carried out using an EcoR I and a Mse I site primer, both without selective bases. .The pre-amplification products were diluted (5 folds) with TE buffer and used as starting material for selective amplification.Then selective amplification was carried out using an EcoR I site primer and a Mse I site primer with three selective bases, respectively. At the end of the selective amplification, the samples were denatured by adding 10u L loading buffer and heated up at 95℃ for 10 min, then cooled on ice immediately. The selective amplified fragments were analyzed by gel electrophoresis. Each sample of 6.5 uL were loaded on 6% acrylamide/bisacrylimade gels. The gels were stained using the AFLP silver-staining protocol, which includes discoloring, rinsing, staining, developing, fixing, and drying.3. Twenty EcoRI/Msel primer pairs showing high level of polymorphism and scorable strong bands were selected out from 64 primer combinations and used for further practical AFLP analysis. One thousand bands were scored, and 88.7% of which (887) were polymorphic. The rates of polymorphic band varied among astringent persimmon cultivars, non-astringent persimmon cultivars, D. lotus, D. olefera, and D. glaucifolia.4. The fingerprints of 120 germplasm of persimmon were established based on AFLP bands using 20 primer combinations. Some of them have unique band patterns, which can be used to their identification.5. The genetic distance standard for classification of persimmon cultivars was determined. Based on the standard, 'Wufengniuxinshi', 'Ganmaokui', and 'Zhouquniuxinshi' are the same cultivar. So are the cultivars of 'shuhuangshi' and 'Huishi', 'Laopige' and 'houquhuoshi', and 'Mopanshi' and 'Maoershi'. These results were as same as the results obtained from morphological analysis.6. According to the dendrogram obtained, t...
|
PDF Full Text Request