Initial Construction Of ISSR-DNA Fingerprinting Database For MengDing GanLu

As traditional and historic tea, MengDing GanLu comes from MengDing mountain, ya’an, Sichuan province, which famous of its unique qualities. The appreciations of many more literary histories are enough to highlight the characteristics of MengDing GanLu. However, with the huge interest in the tea market, the phenomenon that unreal tea are taking place of historic tea becomes more and more serious, leading to unable to identify by consumer. Therefore, identifying information of MengDing GanLu are extremely urgent.In this resrearch, a simple and useful system, which suits for initially construction of MengDing GanLu DNA fingerprint database, had been established by optimization of MengDing GanLu genomic DNA extraction and ISSR-PCR reaction system, primers screening et al. And DNA fingerprint database for 35 MengDing GanLu had been established and pointed out distribution of MengDing GanLu information. The main results were as follows:(1) The optimal ISSR-PCR reaction system of MengDing GanLu was established as follows:30ng template DNA,0.3μmol/L primer,12.5μL 2×Master Mix (0.05units/μl Taq DNA Polymerase; 4mMMgCl2; 0.4mM dNTPs (dATP, dCTP,dGTP, dTTP)) in 25μL system. The optimal amplification was initial denaturation at 94 癈 for 5min, followed by 35 cycles of 45s at 94℃, 1min annealing, and extension of lmin at 72℃, and a final 10min extension at 72 ℃, and saved at12℃.(2) 12 primers of high polymorphism, good reproducibility and clear bands were screened from 65 random primers. Screened primers analyzed the fingerprint of 35 MengDing GanLu sample.The polymorphic bands accounted for 98.5% of the total and each primer amplified17.1 bands on the average, impling rich polymorphic ISSR primers.(3) 35 MengDing GanLu were analyzed using ISSR marker. Using the core primers combination method (R17、R20、R22、R23 and U844 as core primers, R7、R14、R26、R34、 R38、R42、U8.36 for extended aid primers) initially constructed fingerprint dasebase of MengDing GanLu, from which bands were mainly between 250bp-1000bp.(4) The fingerprint information of known MengDing GanLu could be detected and proved that MengDing GanLu DNA fingerprint initial constructed by ISSR marker was feasible.(5) The conlusion of analyzing rapid identification of MengDing GanLu was that primer mixtures could cause missing bands and nonspecific amplification, however, PCR product mixtures were well preserved the information of each PCR product, and the same size bands would increase bright or widen band.