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Dry Atractylodes Lancea(Thunb.) DC.,which is a perennial herbage plant belonging to the compositae,can resist tumor and decrepitude for the famous traditional Chinese native medicine.At present,it is the question that the efficacy of A. Lancea in Maoshan district is far beyond the A.Lancea in other places,as its output decreased in successive years,some merchants adulterated the A.Lancea of other places into the medicinal materials made of A.Lancea,and used it as the high quality A.Lancea.So,A.Lancea is in need of discrimination,restoration and development. The experiment analyzes A.Lancea and Atractylodes Chinensis(DC.) Koidz.with ISSR and RAPD molecular markers in order to provide the basis for A.Lancea identification.A.Lancea and Atractylodes Chinensis(DC.) Koidz 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 was added to extract total DNA,the modified CTAB methods produce high 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 A.Lancea and A.Chinensis.(1):A suitable RAPD-PCR system was established that a total volume of 25μl ISSR-PCR system consisted of 10×buffer 2.5μl,Taq DNA polymerise 1U,Mg2+ 2.0mmol/L,template DNA 50ng,dNTP 0.3mmol/L,primer 0.5μmol/L.(2):A suitable ISSR-PCR system was established that a total volume of 25μl ISSR-PCR system consisted of 10×buffer 2.5μl.Taq DNA polymerise 1U,Mg2+3mmol/L,template DNA 150ng.dNTP 0.5mmol/L.primer 0.2μmol/L.16 primers which could amplified dearly bands and enriched polymorphism were screened out from 102 random primers and were used to analyze with RAPD among A.Lancea and A.Chinensis.As a result it generated 167 bands from 16 A. Lancea genome DNA amplified by 16 ISSR primers,the bands were good repeated and amplified dearly,of which polymorphic bands number was 136,the percentage of polymorphism equaled to 81.44%,and 142 bands from 4 A.Chinensis genome DNA,of which polymorphic bands number was 83,the percentage of polymorphism equaled to 58.45%;Screened 10 ISSR primers from 100 or more,and amplified the 20 genome DNA,then generated 73 bands from 16 A.Lancea genome DNA amplified by 10 ISSR primers,of which polymorphic bands number was 64,the percentage of polymorphism equaled to 87.67%,and 60 bands from 4 A.Chinensis genome DNA,of which polymorphic bands number was 32,the percentage of polymorphism equaled to 53.33%.It was showed by dendrogram generated using the within-group linkage that different individuals of same varieties differed significantly in genetic distance and A. Lancea and A.Chinensis showed a high degree of similarity.Among the 16 RAPD markers,in A.Lancea samples Primer ISSR03 amplified a specific band of 1100bp,which provide a basis for A.Lancea identification;while the 10 screened RAPD markers could not provide a basis for the identification of A. Lancea and A.Chinensis.
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