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【目的】在已知SNP的情况下,找到适合棉花SNP的简便验证方法。【方法】利用四引物扩增突变体系(tetra-primer amplification refractory mutationsystem PCR,Tetra-primer ARMS-PCR)对棉花SNP分型进行验证。【结果】根据深圳华大基因已组装的棉花转录本做为参考,分别将新海21号和新陆中36号的转录组测序样品比对到参考基因,通过SOAPsnp技术检测样品的单核苷酸多态性。棉花海陆杂交亲本的38个单核苷酸多态性位点设计出的66组SNP引物进行验证。通过优化PCR反应体系,反应条件,调整内外引物浓度和PCR体系的优化,从66组引物中扩增出11组引物,得到了良好的分型效果。【结论】四引物扩增受阻突变体系聚合酶链式反应是一种简单快捷而有效的SNP基因分形方法。
【Objective】 In the case of known SNPs, a simple and convenient method to identify cotton SNPs was found. 【Method】 Tetraprase-PCR (Tetra-primer ARMS-PCR) was used to verify the SNP typing of cotton. 【Result】 Based on the assembled cotton transcripts of Shenzhen Huada Gene, the transcriptome sequencing samples of Xinhai 21 and Xinluzhong 36 were compared to the reference genes respectively. The single nucleotide of the samples was detected by SOAPsnp Polymorphism. A total of 66 SNP primers designed by 38 single nucleotide polymorphisms (SNPs) in the parents of cotton sea-land hybrids were validated. By optimizing the PCR reaction system, reaction conditions, adjusting the concentration of the internal and external primers and the optimization of the PCR system, 11 primer sets were amplified from 66 primer sets, and a good typing effect was obtained. 【Conclusion】 Polymerase chain reaction (PCR) with a four-primer amplification-resistance mutant is a simple and efficient SNP gene fractal method.