RESULTS REGARDING CONCENTRATION, PURITY, INTEGRITY OF DNA AND OPTIMIZATION OF SCAR-SSR MULTIPLEX REACTIONS FOR SOME ROMANIAN APPLE VARIETIES

The study was performed to optimize the method of DNA extraction with CTAB, considering reducing working time (non-repeatability of the extraction stage) by obtaining an optimal quantitative concentration to make dilutions and avoid contamination, which would adversely affect the correct and clearly visible amplifications of areas of interest. On the other hand, the multiplexing of SCAR and SSR techniques aimed at reducing reagents consumption and working time. The 48 samples were represented by young (from the top of annual branches) and mature apple leaves harvested in different periods (June and July) from the same year. The leaves come from the apple collections located in two Romanian research units (Research Institute for Fruit Growing Pitesti, Argeș and Research Station for Fruit Growing Voinești, Dâmbovița). DNA was quantified for 48 samples and extracted from young leaves harvested in June and mature leaves, harvested in July. The readings made with the help of the Nanodrop 2000 spectrophotometer showed high concentration values for young leaves and lower concentrations for leaves harvested in July. Evaluation of the DNA concentration for the 48 samples allowed the quantitative/qualitative evaluation and a correlation of the amount of DNA with the stage of development of the leaf. Optimization of SCAR-SSR multiplex reactions for the same DNA concentration (50 ng / μl), led to optimal amplifications for the fragments associated with genes of interest in the SCAR technique, as well as for those amplified by the SSR technique.