洋葱Ms位点多重PCR标记的开发与优化

Development and Optimization of Multiplex PCR-Based Marker of Ms Locus in Onion

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。

Through designing and screening primers on the basis of flanking sequence of Ms locus, a compatible multiplex PCR-based marker （ MK4 ） was obtained , and its reaction system and procedure were optimized.The optimal PCR reaction system consisted of 2.5μL of 10 ×PCR buffer （Mg2＋free）, 4μL of 25 mmol/L MgCl2, 6 μL of 2.5 mmol/L dNTP, 1 μL of DNA template （about 50 ng）, 1 μL of each of 10 mmol/L primer, 0.6μL of 5 U/μL rTaq DNA polymerase , and finally adding ddH 2 O to 25μL.The PCR re-action procedure was initial denaturing at 94℃ for 5 min, followed by 35 cycles of denaturing at 94℃ for 30 s, annealing at 65.4℃for 45 s, extending at 72℃for 1 min, and then extending at 72℃for 10 min in the end.Three genotypes （ MsMs, Msms, msms） of Ms locus could be clearly distinguished by MK 4 based on the optimal reaction conditions and procedure .The reaction was simple and had good stability .