Objective:To optimize and validate the molecular marker of"CMS"triplex PCR in pepper.Methods:The DNA extracted by the modified CTAB method was used as a template to optimize the multiplex PCR molecular marker system related to the three lines of"CMS"in pepper,and the fertility identification of 20 pepper germplasm materials was carried out to verify the accuracy of the optimized multiplex PCR.Results:The optimized 20 µL PCR system included 10 µL of PCR Mix,1 µL of primer SCAR130/140 and 1 µL of CRF-SCAR,1 µL of DNA template,and the insufficient part was supplied by ddH2O.The optimized PCR amplification procedure was pre-denatured at 94 ℃ for 3 min,denaturation at 94 ℃ for 30 s,renaturation at 57.8 ℃ for 30 s,extendation at 72 ℃ for 90 s with 35 cycles,then extended at 72 ℃ for 5 min at last and stored at 4 ℃.The optimized multiplex PCR was used to verify the A,B,and C materials,and the accuracy rates were 85.7％,100％and 80％,respectively.Conclusion:The optimized multiplex PCR reaction system has good repeatability,stability and reliability.
关键词
辣椒/胞质互作雄性不育/多重PCR/分子标记
Key words
Pepper/Cytoplasmic interaction male sterility/Multiplex PCR/Molecular marker
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