Establishment Of DNA Extraction, Purification And Quantification Technique System In Diospyros Spp.

Diospyros spp.,as a perennial woody plant,have high contents of polysaccharides, polyphenols, or other secondary metabolites that can interfere with DNA extraction and purification.In this research,we investigate to extract DNA from most of organs tissue culture, bud, annual phloem, young leaf, flower, sepal, young fruit, matured leaf, matured fruit, old leaf, dried permission ect, in attempt to establish a optimal system of high-quality genomic DNA isolation, purification and quantification that is free of sampling-date and matirials. This will provide a technical basis for the conservation, indentification, estimate and utilization of plant genetic resourse, also giving a widespread application of plant cultivar protection, marker-assisted selection in breeding and many molecular downstream researches. The results were as follows:1. Different organs DNA is extacted by using alkaline lysis method, chlorobenzene extraction, high salt low pH method, improved SDS, improved CTAB, DNA extaction kit. Alkaline lysis method is simple, time-saving, but the qulity is poor and instable.Other five DNA extraction methods had made some certain simplification compared with the original method, D260/OD280 is 1.66-1.78, the DNA quality was still good, can be applied to Polymerase chain reaction.The result shows that simplifying DNA extraction protocol is feasible for PCR which is not stricted with DNA quality.2. Based on CTAB DNA extraction method, some treatments with different sampling, purifying, precipitating were studied to preserve the effect on genomic DNA extraction in some Diospyros spp. The results indicated that as the tissue developed, the yield of DNA decreased. Compared with tender shoots, the amount of DNA from mature leaves was 16.7-18.4%. Among three purification methods, the high salt/water-saturated ether obtained high-quality easily digested DNA with higher yield. In addition, add high concentration salt before organic precipitation is helpful to remove the impurity, could gain higher-quality DNA.