Studies On Growth Characterization And Genomic DNA ISSR Fingerprinting Of Different Pseudopeziza Medicaginis

The objective of this study was to measure and analysis the growth characterizes of 7 Pseudopeziza medicaginis, from different cropping areas in China. And PCR-based ISSR fingerprinting analysis was carried out to one of 6. The results of each independent experiment were respectively described as follows:1. By isolating and cultivating 7 Pseudopeziza medicaginis, come from Beijing, Gansu, Beijiang, Nanjiang, Shanxi, Inner Mongolia and Ningxia. Furthermore, ensure the method of Lixinxishi was optimum for Pseudopeziza medicaginis, and the method of ascospore discharge can not being colony.2. By measuring and analysing the diameter and growth speed, found that Nanjiang strain have advantage, but can not put up significant differences. One of Beijiang, Nanjiang and Inner Mongolia produce ascospores, but nothing of the others. Maybe the condition of produce ascospores has significant differences to different strains. The result of AVONV showed that the Nanjiang and Inner Mongolia put up consistencies on the size of apothecia and ascospores. On the speed of growth and the time of produce colony, the Ningxia strain all have disadvantage with others, and have a remarkable differences.3. By comparing four culture medium of V-8 calcium carbonate, V-8, V-8 calcium carbonate and medic juice and V-8 medic juice, find V-8 medic juice have the best results.4. Compared with DNA quality of three methods showed that Benzyl chloride was the best way, and the result of SDS-CTAB was secondary, and SDS was the worst.5. By distinguishing the Pseudopeziza medicaginis at molecular level, the ISSR-based fingerprinting analysis was carry out by using genomic DNA from 6 strains collected from different areas. The results showed that the optimal ISSR reaction system comprised 100pg/μL template DNA, 0.5mM ISSR primer, 2.0 mM MgCL₂, 0.2mM dNTP, Taq DNA -polymerase 1U, 2.5% formamide deionized and 2μL10×buffer in a total volume of 20μL reaction mixture, with the procedure of one cycle denaturing at 94 for 4min;10 cycles(-1℃/cycle) each involve denaturing at 94℃for 30s, annealing at 58℃for 45s;extending at 72℃for 2min; and 33 cycles involve denaturing 94℃for 30s, annealing at 48℃for 45s;extending at 72℃2min, a final extension at 72℃for 5min after the last cycle; and then remaining at 25℃.