Studies On Genetic Diversity Of36Varieties In Pomegranate(Punica Granatum L.) By SRAP

Pomegranate(Punica granatum L.) which is included in the family of Punicaceae, the genus Punica is deciduous shrub or small trees. Molecular marker which is studied genetic diversity and differentiation is an effective tool. SRAP markers have been widely used in the analysis of genetic diversity for their simplicity, rapidity and stabilization.In this study, the36varieties of pomegranate on the basis of genomic DNAs extraction to complete the test and SRAP analysis on genetic diversity of which is derived from the pomegranate maternal garden of Liangshan Yi Autonomous Prefecture, Huili County in Sichuan Province, the main results showed as follows:1. A good genomic DNA extraction method was found for Pomegranate, which was as follows:the test material choose the pomegranate leaves which are dried by silica gel, used the modified CTAB method, PVP30and Vc in the ground in liquid nitrogen to prevent oxidation of phenolic compounds; cracked the nucleus before joining STE solution, using low-speed centrifugation to collect the precipitate, which can effectively separate the nucleus from polysaccharides, polyphenols and secondary material; ultimately obtained the quality of the pomegranate genomic DNA by phenol-chloroform-isoamyl alcohol mixture extracted2-3times.2. The PCR system for SRAP analysis was as follows:total volume20μl, lμl forward primer,1μl reverse primer,2μl template DNA,1U Taq Polymerase,0.5mmol/L dNTP each,3.0mmol/L Mg2+. SRAP-PCR Procedures:pre-denatured at94℃for5min, denaturation at94℃for lmin, annealing at35℃for lmin, extension at72℃for lmin,5cycles, denaturation at94℃for1min, annealing at50℃for1min, extension at72℃for1min,35cycles, final extension at72℃for5min, remaining at4℃.3. Special amplification bands based on SRAP markers were obtained in36varieties of pomegranate.30pairs of primers producing qualified fragments were selected from90pairs of SRAP primers in order to carry through their genomic DNA for PCR amplification. The results showed that5868loci were amplified totally by30pairs SRAP primers of which4032loci (68.71%) were polymorphic in the species level. DNA fragments were amplified ranging from100bp to2000bp, different primer amplified different amounts of polymorphic SRAP loci, ranging from78to251. The average number of polymorphic loci produced by each pair primer was134.4.4. Using the cluster analysis,36varieties were divided into8groups of similarity coefficient at0.77. Group Ⅰ included2varieties, Group Ⅱ included2varieties, GroupⅢ included3varieties, Group Ⅳ included only one variety, Group Ⅴ included6varieties, Group Ⅶ included5varieties, Group Ⅷ included only one variety, Group Ⅵ included16varieties.