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以本课题组已获得的洋葱Ms位点侧翼序列为基础,设计、筛选引物获得了一个兼容的多重PCR分子标记,并对其反应体系和反应程序进行了优化。优化后的扩增体系:10×PCR buffer(Mg2+free)2.5μL、25 mmol/L MgCl24μL、2.5 mmol/L dNTP 6μL、DNA模板1μL(约50 ng)、10μmol/L引物各1μL、5 U/μL rTaq聚合酶0.6μL、用灭菌双蒸水补齐至25μL;反应程序:94℃预变性5 min;94℃变性30 s,65.4℃退火45 s,72℃延伸1 min,35个循环;最后72℃延伸10 min。优化后的体系和程序可以检测到清晰的目的条带,通过一次PCR反应即可鉴定Ms位点的3种基因型(MsMs、Msms、msms),操作简单,稳定性好。
Based on the flanking sequence of Ms site obtained by our group, we designed and screened the primers to obtain a compatible multiplex PCR marker and optimized its reaction system and reaction program. The optimized amplification system was 2.5 μL of 10 × PCR buffer (Mg2 + free), 24 μL of 25 mmol / L MgCl 2, 6 μL of 2.5 mmol / L dNTP, 1 μL of DNA template (about 50 ng), 1 μL of 10 μ mol / L primer, / μL rTaq polymerase 0.6μL, with sterile double distilled water to make up to 25μL; reaction procedure: 94 ℃ denaturation 5 min; 94 ℃ denaturation 30 s, 65.4 ℃ annealing 45 s, 72 ℃ extension 1 min, 35 cycles ; Last 72 ℃ extend 10 min. The optimized system and program can detect a clear objective band. One genotype PCR reaction can be used to identify three genotypes (MsMs, Msms, msms) of Ms locus, which is easy to operate and has good stability.