[ Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea. [Method] O. euyo- paea genomic DNA was extracted from leaves as the template for optimizati...[ Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea. [Method] O. euyo- paea genomic DNA was extracted from leaves as the template for optimization of ISSR-PCR reaction system by single-factor experiments on the main factors including Mg2＋ , dNTPs, primer concentration and template amount. [ Result ] The optimal ISSR-PCR reaction system for O. euyopaea was obtained, with a total system vol- ume of 20μl containing 1 × Taq buffer, 3.5 mmol/L Mg2＋ , 0.4 mmol/L dNTPs, 1.0 μmol/L primers, 1.0 U of Taq DNA polymerase and 20 ng of DNA tem- plate. The optimal ISSR-PCR reaction program was started with predenaturation at 94 ℃ for 5 min, followed by 40 cycles of denaturation at 94 ℃ for 30 s, annea- ling at 52 - 55 ℃ for 30 s, and extension at 72 ℃ for 2 min ; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR products. PCR products were stored at 4 ℃. Based on the above conditions, 11 primers with high polymorphism, clear amplified bands and good repeatability were selected. [ Conclusion ] This study laid the foundation for further diversity research and species identification of O. euyopaea germplasm展开更多
AFLP(amplified fragment length polymorphism) is a very powerful fingerprinting technology. The key of making variety fingerprints is to select specific powerful primers for each crop. A quick and effective procedure f...AFLP(amplified fragment length polymorphism) is a very powerful fingerprinting technology. The key of making variety fingerprints is to select specific powerful primers for each crop. A quick and effective procedure for selecting AFLP primers for rice variety fingerprinting was established as the following: (1) Choose3 or more group materials that have close genetic relations. (2) Select potential polymorphic primers from primer pairs that are 2 + 2 primer crosses and same at two ends. (3) Recombine the selected potential polymorphic primers and choosing more polymorphic primers. (4) Add one selecting base at one end to become 2 + 3 or 3 + 2 primers and further selecting more polymorphic primers. Some primers were selected with this procedure, such as M21 P87 and M73P17, with which the fingerprints had more polymorphism and high quality.展开更多
基金Supported by Project of Important New Product Development in Yunnan Province ( 2009BB006)
文摘[ Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea. [Method] O. euyo- paea genomic DNA was extracted from leaves as the template for optimization of ISSR-PCR reaction system by single-factor experiments on the main factors including Mg2＋ , dNTPs, primer concentration and template amount. [ Result ] The optimal ISSR-PCR reaction system for O. euyopaea was obtained, with a total system vol- ume of 20μl containing 1 × Taq buffer, 3.5 mmol/L Mg2＋ , 0.4 mmol/L dNTPs, 1.0 μmol/L primers, 1.0 U of Taq DNA polymerase and 20 ng of DNA tem- plate. The optimal ISSR-PCR reaction program was started with predenaturation at 94 ℃ for 5 min, followed by 40 cycles of denaturation at 94 ℃ for 30 s, annea- ling at 52 - 55 ℃ for 30 s, and extension at 72 ℃ for 2 min ; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR products. PCR products were stored at 4 ℃. Based on the above conditions, 11 primers with high polymorphism, clear amplified bands and good repeatability were selected. [ Conclusion ] This study laid the foundation for further diversity research and species identification of O. euyopaea germplasm
文摘AFLP(amplified fragment length polymorphism) is a very powerful fingerprinting technology. The key of making variety fingerprints is to select specific powerful primers for each crop. A quick and effective procedure for selecting AFLP primers for rice variety fingerprinting was established as the following: (1) Choose3 or more group materials that have close genetic relations. (2) Select potential polymorphic primers from primer pairs that are 2 + 2 primer crosses and same at two ends. (3) Recombine the selected potential polymorphic primers and choosing more polymorphic primers. (4) Add one selecting base at one end to become 2 + 3 or 3 + 2 primers and further selecting more polymorphic primers. Some primers were selected with this procedure, such as M21 P87 and M73P17, with which the fingerprints had more polymorphism and high quality.
