| Fructus Corni is the pulp of the ripe fruit of Cornus Officinalis Sieb.et Zucc., which is removed its stones. It can supply the kidney and the liver, astringe essence and stem desertion. It's one of Traditional Chinese Medicines which The Chinese government has made great efforts to develop, mainly used in vertigo, tinnitus, etc. Pharmacological research on Fructus Corni has showed that it can regulate immune function of mice and decrease blood sugar in diabetic mice. So it is widely used in clinical practice, at the same time, it is used as the material of a lot of Chinese patent medicine such as Liuweidihuangwan, so there is a large needs in Chinese Patent medicines'production. The scholars in our country research the different plants of Cornus officinalis on the shape, size, weight and color. The fruit shape of Cornus officinalis can be divided into ellipticform, cylindericform, long cylindericform, short cylindericform, long pear-shape and short pear-shape. The study on Cornus officinalis was concentrate on chemical component and pharmacological action, few study is on efficacy substance of intraspecific variation—genetic resource, as a result, it is not useful for utilization and protection of Cornus officinalis resource.The intraspecific polymorphism of chloroplast DNA makes a difference to the foundation of phylogenetic relationships on the basis of chloroplast DNA ,and has a strong impact on the foundation of systematic evolutionary relationships of the units of classification on the lower level (such as the levels of species). Lacking in the understanding of the intraspecific differences in chloroplast DNA will result in unreliable systematic evolutionary relationships. The conservative property of chloroplast DNA in higher plants is not absolute. Intraspecific variations of chloroplast DNA on different degrees take place within many species. Therefore, it is very important to know or to exactly estimate the intraspecific variations of cpDNA. Our laboratory has set up RAPD frigerprint of Cornus officinalis on a DNA molecuar level, and get the specific sign of SCAR of different cultivar variety and their full sequence of ITS. 130 samples (leaves dried with silica gel) were collected from Henan, Shanxi and Zhejiang. The Chloroplast DNA has been recovered and analyzed by RAPD method. The main results as follows:1. We change the conventional method and make the new methods according with SCAR-PCR. Chloroplast DNA was obtained in amount of 351μg/ml～2049μg/ml from the leaves of Cornus officinalis, which is low in impurity and amplification, tested by agarose gel electrophoresis and UV spectrophotometer.2. The reaction system and the cycle program was tested and compared. (1) Reaction system: an optional reaction system suitable for the assay and usage of RAPD was established. The volume of amplified reaction was 25μl, containing 10×buffer2.5μl, dNTP(2.5mmol/L)3.0μl, Mg2+ (25 mmol/L)3.0μl, primer(2μmol/L)2.5μl, template DNA(0.351μg/μl～2.049μg/μl) 3.0μl, Taq DNA polymerase (5 U/μl)1μl, deionzied water 9.5μl。(2) The reaction program was that 94℃predenaturation for 5 min in the former denature, using 35 cycles, each cycles containing 94℃denaturation for 1min, 35℃annealing for 1min, 72℃extension for 1min, 72℃posextension for 5min in the final extension.3. 15 primers were chosen to be used for 130 samples, for they showed the strong, reproducible amplification and distinct polymorphism from 100 random primers. A total of 57 DNA fragments were amplified from 200 bp～2000 bp, among which 40 bands was polymorphic, accounting for 70.18%,which can tell that the genetic diversity of the 130 samples was good. The genetic similarity correlation was calculated by SPSS 16.0 Dice method, cluster analysis was carried out using Between-groups linkage method and the genetic dendrogram was established. The cluster analysis indicate that, spindleform was to be one group, cylindericform and short cylindericform cluster together, which have the far genetic relationship with other types. Long pear-shape and cylindericform has the close genetic relationship and cluster together with long cylindericform and ellipticform which have the closest genetic relationship. The genetic relationship between the various cultivars can be seen that from the dendrogram, long cylindericform and ellipticform cluster firstly, secondly long pear-shape and short pear-shape cluster together,then the two groups combine, thirdly cylindericform and short cylindericform get together, cluster with the other cultivars finally. The results offer the evidences on cpDNA molecular level for the classification of Cornus Officinalis.4. Specific strap were screen out by RAPD like ST479. The selected PCR products were recovered, cloned and sequenced. Eight differetial primers were designed by using Primer premier 5.0. With the three pairs selected, the differential cultivar Cornus officinalis and the adulterant counterfeit materials were amplified. Then we get some gerenal PCR reaction systems:(1)50μl reaction system : containing, 10×PCR buffer 5μl, dNTP(2.5mM) 6.0μl, Mg2+(25mM) 6.0μl, each primer(10μM) 3.0μl, template 6.0μl, Taq DNA polymerase(5 U/μl) 2.5μl,deionzied water 17.5μl。(2)The reaction program was that 94℃predenaturation for 5 min in the former denaturation; using 40 cycles, each cycles containing 94℃denaturation for 1 min, 45℃(ST479C) or 55℃(ST479E), annealing for 1 min, 72℃extension for 1.5min, 72℃posextension for 5min in the final extension.The results of this experiment reveal that after the SCAR amplification of the cpDNA of the samples of Cornus officinalis, Vitis vinifera Linn., Fructus Crataegi, and Cornus kousa with the SCAR primer ST479C design according to differential belt ST479, Cornus officinalis can produce a single PCR product while the other three samples produce two to four PCR bands, and this can be used as the evidence of identifying Cornus officinalis. The primer ST479E have high selection in Cornus officinalis, for they can bring one PCR band in the seven varieties of Cornus officinalis and show basically the same size of the band with the original RAPD mark, and the graphs reveal that Cornus officinalis, Cornus kousa, Cornus macrophylla, Fructus Crataegi, and Vitis vinifera Linn. have totally different Strip belts,so we can identify the Cornus officinalis and its counterfeits with primer ST479E.
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