Using SRAP Markers To Analyze Genetic Diversity Of Kiwifruit And Cloning The Gene Of GPX And CAPX

Kiwifruit is the plant of Actinidiaceae and China is the main production area. Since different kinds of kiwifruits need different growth environment, it has great significance to study the relationships among varieties to plan the planting areas reasonablely.So far the studies on kiwifruit focused on breeding, resource development, cultivation, pests and diseases control, physiological and biochemical, storage and processing.There is no report on study of kiwifruit by using SRAP mark technique.This study used the leaves of kiwifruit as experimental material, utilized the SRAP mark technique to analyze the genetic diversity of24materials. Two cDNA segments of peroxidase were obtained in the process of scavenging ROS by using RT-PCR, one for GPX and the other for cAPX.The main results are as follows:1.Optimized the SRAP-PCR system of kiwifruit.In the25μL reaction system the optimum concentrations of each components were as follows:Mg2+2.50mmol·U1, dNTPs0.25mmol·L-1, primers0.2μmol·L-1,Taq DNA polymerase1.25U and DNA template150ng. The SRAP-PCR reaction protocol was:initially denaturing at94℃for5min, then pre_amplifying at94℃1min,35℃1min,72℃1min for5cycles, amplifying for33cycles when the annealing temperature was adjusted to53℃, finally extending at72℃for10min.2. Screened17pairs of primer combinations from100pairs, which had rich banding patterns, stable consequences and high polymorphisms.And used these17pairs to analyze the genetic diversity and relationships of24kiwifruit materials, statistics analysis the amplification results.2462bands were detected, and among them2199were polymorphic bands, polymorphism rate was89.32%. Nei’s genetic similarity coefficients were generated and dendrogram was constructed using UPGMA by NTSYS-PC program. The genetic similarity coefficient among24materials ranged from0.54to0.90, average similarity was0.72. Totally speaking, the genetic basis among24materials was relatively narrow. The result of cluster analysis showed that Actinidia eriantha could be separated when genetic similarty coefficient was0.60; these24materials would be clustered into four groups as actinidia chinensis, actinidia deliciosa, actinidia eriantha and one wild type, which indicated the differences between these types. 3. RNA of kiwifruit was extracted from young leaves.The primers of GPX and cAPX were designed by the conserved region,362bp cDNA fragment encoding glutathione peroxidase and561bp cDNA fragment encoding ascorbate peroxidase were obtained by RT-PCR. And bioinformation of these two cDNA sequences such as the ORF (open reading frame), amino acid sequence predicted were performed on NCBI. The obtained sequences were proved to be the target fragments; these results would lay the foundation for further study on full length cDNA of GPX and cAPX.