美国黑核桃SSR反应体系优化

优化SSR-PCR反应体系是黑核桃（Juglans nigra L.）SSR基因鉴定和群体遗传等研究的基础。本研究通过对PCR反应中Mg²⁺浓度、牛血清白蛋白（Bovine Serum Albumin,BSA）浓度、Taq聚合酶用量、dNTPs浓度、引物浓度和模板DNA量的组合以及PCR程序组合试验,确定了黑核桃SSR的最佳反应体系，即在10 μL的PCR反应体系中,含10 ng模板DNA,0.1 mg·mL^-1牛血清蛋白（BSA）,0.25 mmol·L^-1 dNTPs,1.5 mmol·L^-1 Mg²⁺ 1 μL 10X Taq DNA聚合酶反应缓冲液,0.5 U Taq聚合酶,1.0 mmol·L^-1单对引物（0.5 mmol·L^-13对引物）。SSR-PCR反应扩增程序为：94℃变性3 min；93℃变性15 s，50℃或者53.5℃退火1 min，72℃延伸30 s，32个循环；72℃后延伸10 min，置4℃保存。利用此反应体系对黑核桃进行PCR扩增并电泳检测，其结果清晰、稳定、可靠，适合进一步对黑核桃群体遗传、基因型鉴定和分子生态研究。

Optimization of SSR Reaction System in Juglans nigra

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.