ABSTRACT
In order to establish the stable andreliable ISSR-PCR System of Lysimachia christinae, L16 (4(5)) orthogonal design, which based on 7 levels of single factor experiment, were used in this study. The variance analysis was carried out by SPSS 19.0, and 5 main factors affecting the reaction system were optimized in 4 levels. The best annealing temperature was selected by the optimized reaction system. And the stability and reliability of this system was tested by 23 samples from different origins. The results showed that the five factors (DNA template, primer, dNTP, Mg2+ and Taq enzyme) were the most impacts on the amplified results of ISSR-PCR of L. christinae. The order of the influence was: primer > Taq enzyme > DNA template > Mg2+ > dNTP. The optimal system, which was determined by multiple comparison on different levels of each factor, was total volume of 25 microL, including DNA template 60 ng, primer 0.3 micromol x L(-1), dNTP 0.2 mmol x L(-1), Mg2+ 1.8 mmol x L(-1), Taq enzyme 1.25 U. The optimal system was stable and reliable tested by 23 samples from different origins. This study lays the foundation for genetic diversity analysis, fine varieties selection and molecular identification of L. christinae, and provides reference for optimization on ISSR-PCR system of other speciesin future.
Subject(s)
DNA Primers , Genetics , DNA, Plant , Genetics , Drugs, Chinese Herbal , Chemistry , Classification , Microsatellite Repeats , Polymerase Chain Reaction , Methods , Primulaceae , Classification , Genetics , Quality ControlABSTRACT
This paper reported a new method of Armillaria mellea isolation-Gastrodia elata tissue isolating. Compared with normal isolating method-rhizomorph isolating method, it showed that the success rate of new method (78% ) was higher than the rhizomorph isolating method (16% ) , besides this, the new method was easier, and growth characteristic of obtained strain was superior to that obtained from rhizomorph isolating method.