结球甘蓝SSR检测体系的建立及优化

摘，要：以结球甘蓝自交系15R(南宝)和14S（北黑大平头）为试材，建立了甘蓝SSR-PCR反应体系，并从 PCR反应组成、扩增程序等环节对SSR-PCR反应体系进行了优化。即10.00 μL反应体系组成为：1× Buffer(含20.00 mmol·L^-1 MgCl₂)、50.00 μmol·L^-1 dNTPs、0.40 U Taq DNA聚合酶、0.40 μmol·L^-1 SSR引物、1.00 μL DNA(60.00 ng·μL^-1)，加ddH2O至终体积10.00 μL；对基本扩增程序作了改进，适 宜的扩增程序为：94 ℃预变性5 min后进行20个迫降循环，94 ℃变性45 s，60 ℃退火45 s，72 ℃延伸 1 min，每个循环降低0.5 ℃；再进行15个循环，94 ℃变性45 s，55 ℃退火45 s，72 ℃延伸1 min；72 ℃延伸10 min，16 ℃保存。对PCR产物的电泳检测采用聚丙烯酰胺凝胶电泳（银染法）。

Establishment and Optimization of SSR Detection System in Cabbage(Brassica oleracea var.capitata L.)

In this study,it established and optimized SSR detection system including reaction components,amplified program,by using cabbage inbred lines 15R（‘Nanbao’）and 14S(‘Beiheidapingtou’).A stable SSR-PCR system was followed:10.00 μL reaction solution contained 1×Buffer，50.00 μmol·L^-1 dNTPs，0.40 U Taq enzymes，0.40 μmol·L^-1 SSR primers pairs，1.00 μL DNA(60.00 ng·μL^-1).PCR amplifying program was divided into two steps,after one denaturing step of 5 min at 94 ℃,DNA was denatured for 45 s at 94 ℃,the annealing temperature was 60 ℃ and dropped 0.5 ℃ for each cycle,until a final annealing temperature of 50 ℃ was reached.For the last 15 cycles of the amplification,an annealing temperature of 55 ℃ was employed.When all cycles were finished,amplification ended for 10 min at 72 ℃ .The polyacrylamide gel electrophoresis was used in the detection of PCR amplification.