| This study aimed at the identification of 22 sweet orange (Citrus sinensis (L.) Osbeck) samples supplied by Chongqing Fruit Research Institute. And results attained were as followings:(1) A convenient and safe method on extracting the genome DNA from sweet orange, suitable for ISSR-PCR, was set. In this method, repeating phenol: chloroform: octanol wash is not needed, and it is not as time-consuming as other methods. The value of OD260/280 of the extracted DNA through this method is 1.65-1.90;(2) An ISSR-PCR system and amplification program suitable for the lab was set, through trials of the annealing temperature, the concentration of MgCl2 and dNTP, and the system and amplification program were:Each 20-μL amplification reaction mixture consists of 10×PCR buffer, 2.2mmol/L MgCl2, 220μmol/L each of dNTP, 1μmol/L primer, 2% formamide, 1U Taq polymerase (Sangon, Shanghai, China), and 25ng of DNA template;Amplification was performed under the following conditions: 1 cycle of 6 min at 94℃;27-35 cycles of 30s at 94℃, 45s at annealing temperature (dependent on the primers, Touch-down PCR), and 2min at 72℃;a final extension of 7min at 72℃.(3) 13 out of 22 ISSR primers were screened out, and amplification programs were optimized through trials on the annealing temperature and the number of cycles. With these 13 primers, 218 bands were amplified, of which 15 were polymorphic, and the sizes of them were 1003000bp. These 15 polymorphic bands could distinguish 11 samples from others. And the result of this study indicated that all these samples were closely related;(4) Comparing results from the character analysis and the molecular makers, we could find probable relationships between them, but they cannot be confirmed through this study.
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