Optimization Of The Experimental System Of The Torreya Aflp

Based on PCR, AFLP is the abbreviation if amplified length polymorphism. It's a technique of marker, which is connected, with RFLP and PCR. Its principle involves three basic steps: (1) template preparation (2) selective amplification (3) denaturing polyacrylamide gels analysis of AFLP products following by autoradiography or non-autoradiography, AFLP is a novel and strong fingerprint technique of DNA .It is connected with the virtue of RFLP and RAPD, so it has characteristic: high efficiency, promptitude, stability, accuracy. The article compares AFLP with RFLP, RAPD, and SSR. The result demonstrate: AFLP can produce abundant and stable genetic marker, so it has better prospect on classification, variety identification, genetic mapping, breeding and; location of target gene in the forestry.Torreya grandis is an endemic species in china. It has very large economic worth. This study is to constrict molecular genetic linkage map of torreya grandis by AFLP markers and classify the on DNA level .DNA extracted from the mega gametophytes by CTAB and cholonform/isonycohl (24:1). Then precipate DNA by ethyl alcohol. We dissolve DNA in for several days. Then date the consistency on UV-120spectrometer .the average consistency of DNA is 139ug/ul.it is enough to undertake AFLP reaction.In this paper, the influencing factor on AFLP experiment was analyzed, discussed and summarized, such as the template quality, the template concentration, the enzyme restriction system, the component of the enzyme restriction, the time and temperature of the enzyme restiction, the T4 ligase concentration, the component of the adaptor-ligation, the time and temperature of the adaptor-ligation and so on. An optimized protocol especially suitable for Torreya grandis was put forward through a series of experiments, according to the protocol, the AFLPconditon can be fast determined for different material and the AFLP band can be clear obtained.The PCR reaction products are analyzed on 4.5% denaturing polyacrylamide gels. After electrophoresis gels are stained by Silver Stain Plus, and are dried on a gel drier for sequence gels .so we can detect DNA polymorphism, we conclude we should take a measured attitude toward the construction of molecular genetic map of important cultured tree.