DNA Fingerprinting Of The Main Cultivated Variety Of Camellia Oleifera In Fujian Province

Identification and evaluation of germplasm resources was the basis for its effective use. 50 samples of Camellia chekiangoleosa, Camellia oleifera and Camellia meocarpa had been collected from 17 regions in cities of Nanping, Sanming, Longyan, Ningde and Fuzhou in Fujian Province. 11 kinds of phenotypic characteristic had been used to analyse the diversity of the phenotype. The result showed that: as the study of genetic variation using traditional phenotype possessed difficultities and limitations, Molecular markers were deemed to be a effective tool to study genetic diversity and genetic differentiation of Camellia oleifera.By esTab.lishing the optimized AFLP system, the genetic diversity of main cultivars of Camellia oleifera in Fujian Province had been studied, and the DNA fingerprint of these samples had been constructed to facilitate the future identification of germplasm resources. The main results were as follows:1. 11 kinds of phenotypic characteristics of 50 samples had been measured and analyzed. The average coefficient of variation was 34.13 % and the maximum coefficient of variation was fresh fruit weight which accounted for 64.88 %. The index of Leaf shape and fruit shape were small, accounting for 14.27 % and 9.64 % respectively. The results of 11 phenotypic characteristics indicated that there were wide differences in fresh fruit weight between 50 samples and the leaf shape and the fruit shape were relatively sTab.le characters. Variance analysis showed that difference of pericarp thickness was the largest with the F value of 117.77. In the 11 characters observed, the index of fruit diameter, fruit weight, fruit height, leaf area and leaf shape were the main characters. 11 phenotype correlations analyse showed that: five pairs of characters were significantly related while 27 pairs showed significant correlation. Fruit diameter and fruit weight was most closely related, based on which the fruit diameter - fruit weight regression equation had been esTab.lished, that is Y=4.952X2-15.236X+15.831. UPGMA cluster analysis show that 50 samples were devided into 4 groups. The first group, the second group and No.46,No.47 of the third group were Camellia oleifera. There were 13 samples of group three except for No.46 and No.47 were Camellia meocarpa. No.48 was Camellia chekiangoleosa which was a separate group. This clustering was difficult to identify all the samples.2. The complete genomic DNA were successfully extracted from the leaf of Camellia oleifera which were close to mature by method of modified CTAB. method and using dosage of PVP andβ-Mercaptoethanol that had been adjusted. By using restriction enzyme EcoR I and Mse I, the result of double digestion showed that: genomic DNA had been digested into a diffusion-like which illustrated that the method of extracting DNA had applicable to AFLP analysis. This method had overcome the previous restriction that DNA extraction must use leaflet as the material and had broader applicability and usefulness.3. The best AFLP system for Camellia oleifera had been esTab.lished and optimized. The Enzyme system was as follows: amount of DNA was 150 ng,EcoRI enzyme was 1 U, Mse I enzyme was 3 U. Connection system was as follows: enzymed liquid of DNA was 10μl, EcoR I adaptor and Mse I adaptor was 5 pmol, T4-DNA ligase was 7 U. Pre-amplification system was as follows: connected liquid of DNA was 4μl, final concentration of dNTP was 0.2 mmol·L-1, E0+1 was 2 pmol, M0+1 was 1 pmol, final concentration of Mg2+ was 2.5 mmol·L-1, rTaq polymerase was 1.5 U. Pre-amplified products were diluted 60 times and drew 5μl for the next step. Selective system was slightly different from pre-amplification system with 8 pmol of E0+3 and 3 pmol of M0+3. Selective amplified products were tested by 6 % denatured polyacrylamide gel electrophoresis, and then the colour was flagrated by silver staining.4. 50 samples had been amplified by 8 pairs of primers with stronger amplification, good reproducibility, clear banding patterns, even symmetrical distribution which were selected from 64 pairs of primers. 8 pairs of primers had amplified a total of 313 locus. The average number of DNA locus amplified by each primer combination was 39.1 and the polymorphic rate was 48.24% with 151 locus. In the 50 samples, genetic distance between No.49 and No.50 were the smallest (0.0094) and that between No.26 and No.48 were the largest (0.2328). The average of genetic distance was 0.1236. Samples collected in Minhou and Datian possessed the smallest genetic distance, which indicated that there consisted close relationship between the two provenances. As clustering results were consistent with the facts that it is accurate to identify germplasm resourses using AFLP technology.5. The AFLP technology was used for the first time in the DNA fingerprint mapping of main cultivated varieties of Camellia oleifera in Fujian Province. According to various amplified locus, each sample could be distinguished using method of binary classification and identified.The study above revealed the genetic diversity of cultivars of Camellia oleifera in Fujian Province which would accumulate data for conservation of Camellia Germplasm and breeding of fine varieties in Fujian Province. DNA fingerprint constructed were of great value in identifying good species of Camellia oleifera and of importance for utilization of Camellia oleifera cultivars in Fujian Province.