RAPD Analysis Of Emblic(Phyllanthus Emblica L.) Genetic Resources In Fujian Province

In this experiment 34 accessions of Phyllanthus emblica L. genetic resources in Fujian Province were used as materials. The method of leaf DNA extraction was developed; and then the RAPD reaction system was established and optimized; the cultivar (or line) specific RAPD markers were screened; and the genetic relationship and genetic diversity of Phyllanthus emblica L. germplasm in Fujian Province were discussed by RAPD markers and clustering analysis. The obtained results were as follows:I .The development of the method of leaf DNA extraction in emblicPolyphenols, carbohydrate, and pigments etc. were rich in emblic leaves, which were subject to combine with DNA to form insoluble compounds, and which inhibited the activity of Tag enzyme, therefore which affected the PCR. In this study, in order to overcome the disturb of polyphenols effectively, the improved SDS method was developed, in which the fresh and tender leaves were used as the materials and 3% PVP was added in the extraction buffer. By the improved method, the pure and high-quality DNA were extracted from emblic leaves, which was suitable for RAPD amplification.II. The optimization of RAPD reaction system in emblicBased on the common RAPD reaction program and adjusting experiments, the optimized amplification program for emblic RAPD-PCR was: 94? for 240 s; 45 cycles at 94癈 for 60s, 38癈 for 90s and 72癈 for 120s; 72癈 for 600s. The RAPD amplification system was in a 25 ?L reaction mixture containing 0.6mmol ?L-1 Mg2+, 500 ?mol ?L-1 dNTP, 300n mol ?L-1 primer, 25 ng DNA and 1.25 unit Taq DNA polymerse.HI. The analysis of RAPD polymorphic degree in emblic 34 accessions of emblic germplasm in Fujian Province were analyzed by RAPD technique. 259 sites were totally detected by 20 random 10-mer oligo-mucleotide primers, all of which belonged to polymorphic sites. The polymorphic degree was up to 100%. On the average, one primer produced 12.95 sites. The genetic distances among materials were between 0 and 1, among of which the genetic distances were 0.43~1 between cultivars and the wild, and 0.27~0.99 between Putian and Huian emblic resources.IV. The screen of specific RAPD markers of cultivars (or lines) and cultivar434 accessions of Phyllanthus emblica L. germplasm were used for amplification with 20 random 10-mer oligo-nucleotide primers. The results indicated that 47 RAPD characteristic markers were amplified in 23 accessions with 19 primers and 36 common characteristic RAPD markers within two cultivars or lines were amplified in 34 accessions with 17 primers.V. Clustering analysis of RAPD markers in emblic genetic resources in Fujian provinceThe 34 accessions of Fujian emblic germplasm were divided into 2 groups by clustering analysis with ED (Euclidean distance) method-Group of emblic from Huian and Group of emblic from Putian, and 3 groups with Cosine method-the cultivars, the wild from Huian and the wild No.1 from Putian.