RAPD Analysis Of 23 Olive (Olea Europaea L.)Cultivars

Random amplified polymorphic DNA (RAPD) was used to study the classificationand identification of 23 olive (Olea europaea L.) cultivars. Firstly, the method of DNAextraction was modified for olive DNA extraction to acquire high quality olive DNA thatwas used for RAPD analysis. Secondly, RAPD reaction systems were selected forestablishing optimal RAPD ampification system. The primers that were sieved were usedfor sample DNA PCR amplfication. Finally, RAPD fmgerprint maps and experiment datawere counted and analysed to acquire molecule characteristic of olive samples.Phylogenetic relationships of twenty three olive varieties were studied based onphylogenetic tree by use of UPGMA method.The main results of this research are showed as followsâ‘ The improved CTAB method was the best one for the extraction of olive genomicDNA. DNA could be extracted from fresh young leaves and young leaves that werepreserved by silica gel. DNA from young and tender leaves had better purity and the outputwas high: the output was 194-352ng/mg, OD₂₆₀/OD₂₈₀ of DNA was1.82-2.08. Theintegrality of olive genomic DNA was preferable which was detected by agarose gelelectrophoresis. The extracted olive genomic DNA was satisfactory for RAPD analysis.â‘¡Optimal ampification system of RAPD was established. DNA was amplified in 20μL using the reaction mixtures containing 1×PCR buffer, 1.5mmol/L MgCl₂, 0.2mmol/Lof dNTPs, 1U of Taq Polymerase, 0.25μmol/L of primer, 40ng of template DNA. The PCRreactions were performed in a thermal cycler (PTC-100) programmed for 1 cycle of 2 minat 94℃followed by 40 cycles of 45 sec at 94℃, 1 min at 36℃and 2 min at 72℃for denaturing, primer annealing and extension, respectively. The last cycle was followed byincubation for 4 min at 72℃. Amplification products were conserved at 4℃. AmplificationRAPD products were analysed by gel electrophoresis run in 1.5ï¼…agarose in 0.5×TBEbuffer.â‘¢The 11 primers which produced the highest number of polymorphic bands andshowed consistent and reproducible results were sieved from eighty 10bp primers usingoptimal ampification system. All the reactions were performed three times.â‘£Eleven 10bp primers which were selected from eighty arbitrary primers wereapplied to the amplification. Total 127 bands were produced, in which 78 bands (61.4ï¼…)were polymorphic. The average numbers of DNA bands and polymorphic DNA bandsamplified by each primer were 11.55 and 7.09 respectively. Amplification DNA segmentsize was between 300bp and 2000bp, and the many segments centralized between 500bpand 1500bp. A dendrogram showing genetic relationships was constructed through anunweighted pair-group method (UPGMA) and the 23 cultivars were clustered into 2 maingroups.⑤There were specific bands in four cultivars, ie Arbequina S₁₀₁₁-600bp, KaliniotS₁₀₁₉-1400bp and S₁₀₂₅-2400bp, Frantoio S₁₀₂₅-400bp, Coratina S₁₀₁₁-650bp, and thesebands can be used in olive germplasm identification.