羽衣甘蓝SRAP体系建立与优化

The Optimization of SRAP-PCR Amplification Program and System in Kale

以羽衣甘蓝基因组DNA为模板,对相关序列扩增多态性聚合酶链式反应(SRAPPCR)体系的各影响因子进行了单因素梯度设置,并优化了反应程序,筛选和建立了可扩增多态性高、重复性好、带型清晰的最佳SRAP反应体系和程序。结果表明:羽衣甘蓝最佳SRAP-PCR反应体系总体积10μL,包含DNA模板50ng,1×buffer,dNTPs 0.20mmol/L,Taq酶1U,引物各0.60μmol/L。羽衣甘蓝最佳SRAP-PCR反应程序为94℃预变性5min,94℃变性30s,35℃退火30s,72℃延伸30s,5个循环;94℃变性30s,50℃退火30s,72℃延伸30s,35个循环;72℃终延伸7min,4℃保存。经22个羽衣甘蓝F2群体单株对上述优化的反应体系和程序进行验证,均获得了多态性丰富、条带清晰的扩增图谱,表明该程序和体系能很好地满足羽衣甘蓝基因组SRAP扩增要求。

Taking the genomic DNA extracted from kale leaves as template, each influencing factor of amplified polymorphism polymerase chain reaction （SRAP-PCR） related to the sequence was set by the single factor gradient and the program was optimized, the SRAP which were high polymorphism, good repeatability, clear were screened. The results showed that the optimized protocol was as follows：a total volume of 10μL containing 50 ng genomic DNA, 1 X buffer, 0. 20 mmol/L dNTPs, 1 U of Taq DNA polymerase and 0. 60 μmol/L primers. Protocol run under the following conditions：predenaturing at 94℃ for 5 rain,then denatured at 94℃ for 30 s,annealed at 35℃ for 30 s,and an extension at 72℃ for 30 s for 5 times,denatured at 94℃ for 30 s and annealed at 50℃ for 30 s,and an extension at 72℃ for 30 s for 35 times,extension at 72℃ for 7 min at last, then kept at 4℃. The above optimal SRAP-PCR reaction system and amplification procedure were checked by 22 of F2 individuals. The system and procedure could be used in genomic DNA SRAP-PCR amplification in kale.