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目的:利用单核苷酸多态性(SNP)分子标记建立多重PCR体系,实现人参品种大马牙的分子鉴定。方法:设计内含子侧翼引物,扩增不同人参品种的线粒体Cytochrome coxidase subunitⅡ(coxⅡ)基因第二个内含子核苷酸序列,通过多重序列比对,发掘大马牙的单核苷酸多态性位点(SNP)。根据大马牙的特异SNP位点,设计其等位基因特异性引物,并建立鉴定大马牙的多重PCR体系。结果:在多重PCR体系中,只有大马牙产生了410 bp的特异性条带。将条带切胶回收并经克隆测序验证,该条带确实由大马牙特异性引物Da F和Coxl R扩增得出。结论:本研究建立的多重PCR体系可有效地实现大马牙的快速鉴定,并有望作为大马牙鉴定和田间纯化的一种有效的技术手段。
OBJECTIVE: To establish a multiplex PCR system using single nucleotide polymorphism (SNP) molecular markers to realize the molecular identification of gynura spp. Methods: Intron flanking primers were designed to amplify the nucleotide sequence of the second intron of Cytochrome coxidase subunitⅡ (mitox1) from different ginseng cultivars. Multiple nucleotide sequences Sexual loci (SNPs). According to the specific SNP locus of Maxima, the allele-specific primers were designed and the multiplex PCR system for identification of Larvae was established. Results: In a multiplex PCR system, only 410 bp specific bands were produced in the maxima. The bands were excised and verified by cloning and sequencing. The bands were indeed amplified by Da F and Coxl R, which are specific for the Arabidopsis thaliana. CONCLUSION: The multiplex PCR system established in this study can effectively realize the rapid identification of the maxima and is expected to be an effective technique for the identification and field purification of the maxima.