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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。
Using Kale genomic DNA as a template, single-factor gradient was set for each influencing factor of the related sequence amplified polymorphic DNA polymerase chain reaction (SRAPPCR) system, and the reaction program was optimized. The scalable and scalable High-level, good reproducibility, with the best type SRAP response system and procedures. The results showed that the optimum volume of SRAP-PCR reaction was 10μL, including 50ng DNA template, 1 × buffer, 0.20mmol / L dNTPs, 1U Taq DNA polymerase and 0.60μmol / L primers. The optimal SRAP-PCR reaction program of kale was 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 35 ℃ for 30s, extension at 72 ℃ for 30s for 5 cycles, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles; 72 ℃ final extension 7min, 4 ℃ preservation. The F2 plants of 22 strains of kale were validated by the above optimized reaction system and procedure, and all amplified polymorphic bands with clear bands showed that this program and system could well meet the SRAP Amplification requirements.