Establishment Of Several Molecular Markers And Analysis Of Genetic Relationships In Diospyros Linn.

Japanese persimmon (Diospyros kaki Thunb.) belongs to the Diospyros genus, Ebenaceae Family. China is one of the originating and distributing centres, there are abundant precious genetic resources in China. But the varieties, distribution, origin, evolvement and genetic relationships of Diospyros spp. are unclear for a long time. This study firstly developed the SSR molecular marker from the genus of Diospyros. Then, SSR, SRAP, IRAP and REMAP molecular markers were employed to evaluate the genetic relationships of Diospyros spp. including Diospyros kaki Thunb.; D. Lotus L.; D. glaucifolia Metc.; D.oleifera Cheng.; Jinzaoshi; D.rhombifolia Herosl.; D.virginian L. to elucidate the genetic relationships of cultivars and species of the Diospyros spp. at the DNA molecular level and provide the basis for the scientific and efficient utilization of the persimmon germplasm. The results were as follows:1. SSR primers were developed based on repeated sequences in the public nucleic acids sequences database. A total of 98 sequences of Diospyros Linn., were examined in GenBank, EMBLand DDBJ. Seven SSR sequences were obtained which were all used to design SSR primers. The optimal SSR-PCR system were established2. SSR primers developed using ISSR-suppression PCR technique. Suppression PCR primers were designed to conduct genome walking after eight ISSR primers amplified in genomic DNA. Of the 12 obtained SSR primers, three were monomorphic and the remaining nine showed high polymorphisms. Nineteen sequences were registered in GenBank.3. Six SSR primers were developed using biotinylated (GA)10 and streptavidin-coated magnetic beads. Six sequences were registered in GenBank.4. Eighteen SSR primer pairs were selected to evaluate the genetic relationships of Diospyros spp. from the developed primers. The dendrogram was constructed from the agarose gel data, while the information of alleles was from PAGE gel. Amplification was successful with the 18 SSR markers assayed, detected a total of 158 bands, ranging from 5 to 20 on agarose gel. The alleles collectively yielded unique genotypes for each of the 30 persimmon genotypes, which enabled the unambiguous discrimination of the 30 persimmon genotypes included in this study. The number of alleles per primer ranged...