Optimization Of SSR Reaction System For Genetic Diverdity Analysis Of Jatropha Curcas

Jatropha curcas L., which was categorized to the Euphorbiaceae Jatropha, is deciduous shrubs or small trees, with the height of3-4meters. Since it is native to Central America, it is mainly distributed in tropical and subtropical areas, and arid and hot valley with low rainfall. Wildtype and semicultivated varieties of Jatropha can grow in hot and dry climate under barren soil conditions.Due to the energy crisis countries in the21st century, the current researches focus on biomass energy for the energy strategy of a country, as a new safe, efficient, clean renewable energy resources. Jatropha curcas previously used as a medicinal plant cultivation mostly, is highly concerned by scientists all over the world as a new biomass energy plant. Earlier studies were focused on the research field of plant physiological and biochemical, pharmacology and toxicology, few researches were reported in its biological genetic evolution, evaluation of germplasm resources and genetic breeding.Simple Sequence Repeat (SSR) was popularly applied to analyze germplasm resources. We screened25pairs of high polymorphism, stability primers from120SSR primers, using genomic DNA of26Jatropha curcas as clonal materials, for evaluation of the genetic diversity and genetic relationship of these materials and identification species. This research finally obtained the following conclusions:(1) CTAB method with modification can extract qualified genomic DNA of26Jatropha curcas clonal materials which have good integrity, high purity as a molecular marker of SSR template.(2) Optimum reaction system of SSR-PCR in Jatropha curcas:10×PCR buffer2μL, DNA template100ng, dNTPs0.2mmol/L, Taq DNA polymerase100U/mL, primers0.6μmol/L, Mg2+1.5mmol/L, in the total volume of20μL. Suitable amplification program: pre-degenerated at94for3min; denatured at94for1min, annealed at50-55for1min, extended at7294for2min,35cycles, extended at72for the last10min.(3) In120pairs of primers,87pairs of primers could amplify clear bands, of which only25pairs had polymorphism, so, the polymorphic rate was28.7%. The total of60bands had polymorphism in129identified bands, resulting in46.5%of polymorphic bands.(4) UPGMA cluster analysis and principal component analysis showed that the genetic similarity coefficient of26Jatropha curcas clones material were between0.705to0.992, leading to the average similarity coefficient of0.875. By cluster dendrogram, in the genetic similarity coefficient of0.853,26samples of Jatropha curcas materials were divided into5groups based on the materials numbers:the first group included the No.1,2,6,9,10,17,18,8,11,7,24,4,14,12,3,19,13,26,15, the second group of number21, the third group consisted of No.5,20,16the fourth group of number25, the five group were22and23using the result principal component analysis(PCA). So far, this research revealed that the genetic diversity of26Jatropha curcas clones germplasm resources was smaller, and showed that SSR markers could analyze the genetic polymorphism of Jatropha curcas germplasm resources in some extent.(5)6SSR characteristic bands that distributed in3Jatropha curcas clones, can be used as the basis of identification of varieties of Jatropha curcas.