Molecular Genetic Diversity Of Astragalus Sinicus.L Germplasm And Development Of SSR (Simple Sequence Repeat,SSR) Markers

Astragalus sinicus.L belongs Legume Astragalus annual of herbaceous plants,which is distributed mostly in southern china,middle and lower reaches of YangtzeRiver,Huang-Huai River basin,The United States of America,South Korea,Japan andother countries and also known as Qiao Yao,Clove,Cao Zi,ect. It as green manureto increase the number of soil microorganism,adjust the soil physical and chemicalproperties,promote soil organic carbon accumulation,reduce CO2emissions whichplays a crucial important role in the promotion of low carbon agriculture and organicagriculture,at present.So,protection and development of germplasm resources ofChinese milk vetch has great value.Eleven populations of Astragalus sinicus.L whichcame from Fu Jian,JiangXi,Ning Bo,An hui,were used in the experiment.Thematerials were provided by the Institute of soil and Fertilizer,Fujian Academy ofAgricultural Sciences. Optimized primers and development of SSR primers byISSR-Suppression-PCR are used analysis of genetic diversity of Astragalus sinicus.Lgermplasm resources The main contents and result of this study are as follows.1, The factors that affecting the ISSR(inter-simple sequence repeat)result ofAstragalus sinicus.L. We studied and selected15ISSR primers which can amplifybands from the DNA of Astragalus sinicus.L.and the15ISSR primers all from theISSR primers published by Columbia University. By setting the temperaturegradient,the available primers'best annealing temperature are explored.The effect ofthe five main reaction elements(Mg²⁺,TaqDNA polymerase,dNTP, template andprimer)on ISSR-PCR were all optimized by combining single factor experiments andorthogonal test method.then we establish the best ISSR-PCR reaction system.Thebest ISSR-PCR reaction system of Astragalus sinicus.L is25μl reaction systemcontain the DNA template50.00ng,Mg²⁺concentration2.00mmol/L,Taq enzymedosages1.0U, dNTPs concentration is0.25mmol/L,primers0.20μmol/L,2.5μL10×buffer. The selected ISSR primers and annealing temperature is widespread andrepresentative in Astragalus sinicusl ISSR-PCR marker research and the result couldprovide the basis for the analysis of diversity, protection;exploitation of SSR primersof Astragalus sinicusl.2.The selection of four ISSR were used to develope eight pairs of Astragalus sinicus.l polymorphism of SSR primers.The matrixes of genetic distance wasestablished on SSR fingerprint.The dendrogram shows0.4as the dividing line can be11kinds of Astragalus sinicusl varieties divided into categories,At the same time,canalso be see8487111and other varieties of kinship are obviously different. respectivelyThe results of UPGMA cluster analysis based on production ISSR and SSRfingerprint had the approximate trend,but not the same.In summary,ISSR and SSR molecular markers technic provide a scientific basisfor the analysis and identification of Astragalus sinicusl. Germplasm resourcesgenetic relationships among the country, and developing innovative work of speciesprotection and breeding.