关键词: 黑核桃, 微卫星, 反应体系, 群体遗传

Abstract: The optimization of the SSR-PCR (simple sequence repeat-polymerase chain reaction) reaction system is an important basic protocol when SSRs are used for pedigree construction, genotyping or population genetics research in black walnut (Juglans nigra L.). We systematically tested the concentrations of Taq DNA polymerase, BSA, dNTPs, Mg²⁺, primers, and template DNA concentration in the PCR reaction to determine the optimal reaction system. The results indicated that the optimal SSR-PCR reaction conditions for black walnut (J.nigra L.) included a total volume of 10 μL containing 1 μL of 10 ng·μL^-1 DNA, 1 μL of 10 X Taq DNA polymerase reaction buffer (1.5 mmol·L^-1 Mg²⁺), 1.25 μL of 200 mmol·L^-1 dNTPs (0.3 mmol·L^-1), 1 μL of 1 mg·mL^-1 BSA (Bovine Serum Albumin, 0.1 mg·mL^-1), 1.0 μL of 10 μmol·L^-1 each primer (when using one pair primer in each PCR reaction) or 0.5 μL of 10 μmol·L^-1 each primer (when using three pairs primer in each PCR reaction), 0.5 units Taq polymerase, and 4.5 μL sterilized distilled water. The thermal cycling conditions were as follows: denaturation 3 min at 94℃; 32 cycles of 15 s at 93℃, 1 min at the annealing temperature for the primer, 30 s at 72℃; and a final extension of 10 min at 72℃ at the end of the amplification. The results showed that this SSR-PCR protocol resulted in clear, reproducible results suitable for the analysis of population genetics, genotyping, and for molecular ecology.

Key words: Black walnut, microsatellite, reaction system, population genetics