Application Of Rhizoma Atractylodis Macroccphala Identification By RAPD And ISSR

Dry rhizome of Rhizoma atractylodis macrocephala (Atractylodes macrocephala Koidz.) belonging to the compositae can resist tumor and decrepitude for the famous traditional Chinese native medicine. At present it is the question that YUZHU and DONGZHU belonging to Rhizoma atractylodis macrocephala is at the combination and in urgent danger the condition. YUZHU is in need of the discrimination, restoration and the development, in which the discrimination is a key. The experiment analyze YU ZHU and DONG ZHU with ISSR and RAPD molecular markers in order to provide the basis for Rhizoma atractylodis macrocephala identification.YU ZHU and DONG ZHU in the leaf include the massive phenol which is oxidized extremely easily in the genome DNA extraction process . As soon as phenol oxidized combine to the genome DNA, concentration and purity of Genomic DNA will be affected.Based on the conventional CTAB methods, an efficient procedure was developed. The major improvement included elimination a lot of secondary metabolites such as polyphenols etc, and collection of nuclei before free DNA.Then CTAB-free buffer [100mmol/L Tris-HC1 (pH 8.0); 50 mmol/L EDTA(pH 8.0): 700 mmol/LNaCl; 2%PVP (W/V): 2%β-mer (V/V) ]was added to extract total DNA. the modified CTAB methods produce high quality DNA compared with conventional CTAB methods.The orthogonal design was used to optimize RAPD and ISSR reaction system in order to establish a suitable system for YUZHU and DONGZHU: A suitable RAPD-PCR system was established that a total volume of 25μl ISSR-PCR system consisted of 10×buffer 2.5μl, Taq DNA polymerase 1.25U, Mg²⁺ 2.5mmol/L. template DNA 50ng. dNTP 0.2mmol/L, primer 0.5μmol/L; A suitable ISSR-PCR system was established that a total volume of 25μl ISSR-PCR system consisted of lOxbuffer 2.5μl, Taq DNA polymerase 0.75U, Mg²⁺3mmol/L, template DNA 200ng, dNTP 0.25mmol/L, primer 0.5μmol/L.15 primers of 10 base pair were screened out from 72 random primers and were used to analyze with RAPD among 49 accessions .which generated 112 bands,of which polymorphic bands number was 94,the percentage of polymorphism, equaled to 83.9%;10 primers,which could amplified clearly bands,weres screened out from 100 ISSR primers.As a result,114 bands weres generated, of which polymorphic bands number was 102, the percentage of polymorphism was 89.5%.The result indicated that ISSR is more effective than RAPD.It was showed by dendrogram generated using the within-group linkage that YUZHU intermix with DONGZHU;different individuals of same varieties differed significantly in genetic distance and YUZHU and DONGZHU showed a high degree of similarity .YUZHU and DONGZHU could not be distinguished by RAPD markers; in YUZHU samples Primer ISSR5 amplified a specific band of 375bp , which provide a basis of Rhizoma atractylodis macrocephala and DONGZHU identification.