Establishment Of SSR Reaction System And Analysis Of Genetic Diversity Of Apple Cultivars

Apple is one of important fruit tree in the world, but traditional genetics and breeding costs more time, labor and money due to apple's long juvenile period and life cycle, the large plant and self-incompatibility so on. DNA molecular markers were widely developed and applied to various aspects of research in apple with molecular biology booming in recent years, so it was possible to identify apple cultivars quickly. Especially SSR marker is highly polymorphic, codominant, well-repeated and convenient. In this paper, SSR reaction system of apple was established and the genetic diversity of 42 apple cultivars was analyzed by using SSR marker in order to offer basis for germplasm resources identification , cultivars relationship and plant breeder's rights protection. The main results from this study are as follows:(1) The factors which affected on the SSR results of apple were studied. We had set up the optimizing SSR reaction system of apple: each 20μL amplification reaction solution was comprised 2.5mmol·L^-1 Mg²⁺, 0.8umol·L^-1 primer, 0.10mmolL'dNTP, 1UTaq polymerase, 15³0ng template DNA and the optimal annealing temperature of primers were 48.5⁵2.0℃. The amplification program: a pre- denaturalization step of 4 min at 94℃ for one cycle followed by 30 cycles of denaturalization 1min at 94℃, annealing 1min at 48.5⁵2℃, extension lmin at 72℃ and 5min at 72℃ as final extension step. The result of PCR amplification showed that the PCR system had good and stability.(2) 42 apple cultivars were amplified by 12 pairs of SSR primers .165 bands were amplified and polymorphic percentage of amplified bands was 54.88%. The number of amplified bands per primer pair ranged from 5 to 24, with an average of 13.6. Based on the SSR results, calculation of the genetic similarity showed that the gene differentiation was not remarkable among cultivars. Based on the genetic distance and the UPGMA cluster, all the providing cultivars (lines) except 'Fuji' bud mutation lines could be identified from each other and classified into four groups at the genetic distance of 0.800. Majority closely related cultivars (lines) according to pedigree were grouped together. SSR markers could identify cultivars (lines) that were developed through conventional hybridization breeding method and study the genetic diversity of apple.(3) SSR fragments of 12 pairs of primers were detected by polyacrylamide gel (8%), so a genotype map of providing cultivars at a group of SSR loci was obtained. It would lay out a foundation for further genotype database, also indicating that SSR markers were suitable for variety identification and plant breeder's rights protection.