ISSR Markers And Genetic Diversity Analysis Of Japanese Apricot (Prunus Mume Sieb. Et Zucc.)

Japanese apricot (Prunus mume Sieb. et Zucc.) which belongs to the genus of Prunus L. in Rosaceae was originated in southern China and has high economic value. Japanese apricots was an important fruit tree which have been exported abroad over half the total output the main producing areas in Guangdong in recent years. Japanese apricots are rich in genetic resources and cultivated with a long history, but their morphological characters are very similar. It is a very serious phenomenon that different cultivars have the same name in production. There is much confusion and difficulty in identifying the cultivars. In order to preserve and utilize the germplasm better, it is necessary to study their genetic diversity by ISSR molecular markers.1 Study on genomic DNA extraction from recalcitrant Prunus mume Sieb. et Zucc.The modified CTAB method has been developed to extract the high quality genomic DNA from the recalcitrant fruit tree Japanese apricot (Prunus mume Sieb. et Zucc.) which contains abundant polysaccharide, polyphenol, organic acid and some other secondary metabolites. Some measures were taken to eliminate them including a proper amount of PVP and 2 %β-mercaptoethanol were added during grinding to prevent oxidation of phenolic compounds; using high concentrate of CTAB/ NaCl solution to separate polysaccharides. The results of ratio of A₂₆₀ and A₂₈₀ , electrophoresis and amplification suggested that the modified CTAB method could obtain higher quality genomic DNA with better purity and integrity compared with modified SDS while the conventional CTAB method is not suitable for the DNA extraction from Japanese apricot.2 Establishment of an ISSR reaction system in Japanese apricot (Prunus mume Sieb. et Zucc.)Based on the high quality genomic DNA extracted by the modified CTAB method, five essential factors that might affect the results of ISSR were compared by orthogonal design and single factor test. Comprehensive results of the two methods, a suitable ISSR-PCR reaction system (20 uL) was established, i. e. 2 uL 10×buffer, 2.5 mmol/L Mg²⁺, 0.2 mmol/L dNTPs, 0.32μmol/L primer, 20-80 ng template DNA and 1 unit of DNA polymerase. The parameters were: 5 min at 94℃for initial activation step, followed by 35 cycles of 45 s at 94℃, 45s at annealing temperature 47.1℃-54.3℃and a 2 min extension at 72℃. At the end, amplified products were elongated for 7 min at 72℃and preserved at 4℃.3 The analysis of genetic diversity of Japanese apricot(1) The polymorphism analysis of accessions The genetic diversity of Japanese apricot were studied by ISSR. 10 primers were screened from 51 arbitrary ISSR primers. A total of 120 DNA fragments ranging from 0.2-3.0kb were amplified, among which 98 (81.67 %) were polymorphic, using these 10 primers. The average number of DNA band produced by each prime was 12. The polymorphic band difference was very significant among different primers. The average number of DNA polymorphic band produced by each prime was 9.8.(2) The cluster analysis of accessions According to the result of PCR, the genetic similarity analysis for 39 cultivars was calculated by the software of NTSYS pc - 2.1e. The result of genetic similarity analysis showed that the Jaccard coefficient ranged from 0.5263-0.9910. A DNA molecular dendrogram was established for 39 cultivars based on UPGMA cluster analysis. When the similarity coefficient value was 0.65, the 39 Japanese apricot cultivars were divided into 3 groups, and 34 cultivars of the third group (III) were further divided into 5 subgroups, as was fundamentally accorded with the traditional classification base on fruit-ripening color. There was no obvious difference in geographic relationship among the clustering results. The study indicated that ISSR produced high polymorphism on Japanese apricot. ISSR analysis can be used for the molecular evaluation and studies of genetic diversity of Japanese apricot germplasm.