| China is abundant in Codonopsis germplasm resources. But there's little investigation work of Codonopsis resources had been done and due to a great many of introduction planting among different areas, it caused the code of germplasm resources and genetic relationship confused that makes the researching,conserving,appraising,utilizing and innovating of new Codonopsis ermplasm resources much difficult. The research adopted the technology of AFLP (Amplified Fragment Length Polymorphism), and to construct stable optimized structure, and then based on it selected 2 pairs of clear amplified band,high polymorphic primer combinations, established 12 fingerprinting pattern of Codonopsis samples, analyzed the genetic polymorphism of them, then the work of identifying,analyzing genetic relationship,establishing high density fingerprinting pattern and constructing of core germplasm of Codonopsis should be based on it. The main result shown as follows:1. In accordance with the characteristic of high content of polysaccharide and protein in organize of Codonopsis, improved CTAB method was adopted, and eliminate polysaccharide and other impurities, then selected the best efficient method which fitted for AFLP analysis of extracting genomic DNA of Codonopsis. The result showed that to add 2%CTAB,175mmolL-1 sorbierite and 6.25gL-1 sodium sulfite in DNA extracting solution, then the extracted genomic DNA of Codonopsis are with high purity,basically no degradation and fitted for AFLP analysis.2. Through the system study on AFLP reaction system of Codonopsis, established optimized system fitted for AFLP analysis. The overall volume of 15uL digestion system contained purified DNA300ng,EcoRI 15U,EcoRI Buffer 2uL,first digested 7h by EcoRI in 37℃, then added MseI 5U, digested 5h in 65℃, kept constant temperature 20 minutes in 80℃, inactivated the activity of restriction enzyme, then put the eppendorf on the ice, centrifuge briefly and collected the products.of digestion after cooling down.The linked system contained: 5uL DNA of digestion, MseIadaptor(10pmol/uL) 5uL,EcoRI adaptor (10pmol/uL)1uL,T4DNAligase0.5uL,T4DNAligasebuffer2.0uL,ddH2O6.5uL,bed blending, centrifuge briefly,then linked 7h in low temperature water bath in 22℃, centrifuge briefly and collect products of link.Take 1uL linked products used for pre-amplification.The pre-amplified produce was diluted 50 times with TE buffer and used for selective amplification.After the selective amplification,the Codonopsis samples were denatured by adding 5 uL Loading buffer and heated up in 95℃for 5min,then cooled down on ice immediately.Then conserved in -20℃for standby.Took 4uL denatured PCR reaction products to electrophoresis on 6% denatured polyacrylamide gels and then stained by silver.3. In the process of the AFLP analysis, the technology of silver staining was used instead of radioactive isotope, because it avoided the harm of the radioactive isotope, so experiments operation are more convenient and safe ,and it shortened the periods of the experiments. The factors of affecting the effects of silver staining,such as operation methods and reagent of silver staining also have been discussed.4. In the experiment, two primers been selected can amplify 188 bands, each primers can amplify 94 bands and 173 of 188 amplified bands were polymorphic bands, which acccouts for 92.02%.To cluster part of Codonopsis germplasm resources by UPGMA in genetic identity and genetic distance,the results showed that: Codonopsis samples could be classified into three clusters at 0.63 level of genetic distance. There are seven Codonopsis samples from different places in the first cluster, among which the Codonopsis pilosula (Franch.)Nannf from Chengguan town and Shouyang town, Longxi county in Gansu province have the closest genetic distance that is 0.2392.There are four Codonopsis samples in the second cluster and only one Codonopsis sample in the third cluster, namely, C.convolvulacea from Honghe town in Yunnan province.
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