Inter-Simple Sequence Repeat (ISSR) Analysis In Freesia Refracta

Freesia refracta, one of most famous cutting flowers of the world,has high ornamental and economic values. The genetic diversity and relationships of 12 freesia varieties were investigated with ISSR (inter-simple sequence repeat) to provide the theoretical base for the breeding and variety protection of F. refracta.Firstly, the method of DNA extraction from F. refracta was optimized. On the basis of CTAB method,the ground samples were washed with dd-H2O to elute the phenolic compounds before adding 2 x CTAB extraction solution containing 0.0125mol/L borax. And the polysaccharides were removed by adding 0.35 volume of anhydrous ethanol before phenol/ chloroform/ isopropyl alcohol treatment and supplementing 0.5 volume of 5 mol/L NaCl before DNA deposited by 2 volume of anhydrous alcohol. The quality of DNA extracted from different tissues of F. refracta was compared to decide the opimum DNA extraction organ. The results indicated that the tender leaf was the best choice. Through the optimized method, the high quality F. refracta genomic DNA, using as the template for PCR, was obtained.Secondly, the ISSR-PCR system using for F. refracta molecular analyses was optimized. The system volume was chose as 25μL after comparing the influence of different system volume on PCR results. Then the impacts of Mg2+ concentration, dNTPs concentration, DNA template usage, Taq DNA polymerase usage and primer usage were studied through orthogonal tests L16(45). The optimized system for F. refracta ISSR-PCR analysis contained that, in 25μL volume, 1×Taq buffer (10mmol/L Tris.HCl,pH 9.0,50mmol/L KCl,0.1% Triton X-100),2.0mmol/L MgCl2,0.06U/μL Taq DNA polymerase,4ng/μL DNA template,0.4μmol/L primer,0.6mmol/L dNTPs. The optimum annealing temperature of different primers was decided by gradient temperature PCR.Finally, ISSR-PCR molecular analyses of 12 F. refracta varieties were investigated with the optimized system. The data were computed with NTSYSpc (2.1), and the UPGMA dendrogram for 12 species of F. Refracta on Nei's genetic distance was obtained. According to the results, the 12 varieties were divided into 4 groups, the Baise variety was belong to the group 1, the Dahongchong to the group 2, the yellow clolor and red color varieties to the group 3, and the 3 purple varieties were the group 4. And the results also demonstrated that the genetic background of 12 varities was relatively simple, and their genetic base was relatively narrow.