大豆小片段法HRM基因分型体系优化

Optimization of small amplicon of HRM genotyping system in soybean

应用高分辨率熔解曲线（high resolution melting curve,HRM）技术进行基因分型简单有效,灵敏度高、特异性强。优化并建立基于HRM的大豆SNP基因分型体系,是准确进行SNP研究的前提和基础。本研究利用添加内标后的小片段法进行SNP基因分型体系优化研究,结果表明在设计SNP引物时,最好使其PCR产物长度为50～80bp,熔解温度在72～82℃,Tm值介于55～62℃之间;10μLPCR反应体系中应包含25ngDNA模板,0.6pmolSNP引物,1μLLCGreen染料;PCR反应后,应在各点样孔分别加入3pmol的高、低温内标,然后进行变性处理和HRM分析。同时利用建立的基因分型体系对重组自交系群体（RIL）进行了基因分型检测,能完全将其分成亲本的两种基因型,提高了SNP基因分型的准确性和效率。本研究建立的SNP基因分型体系为今后进行大豆SNP标记开发、高密度遗传图谱构建、QTL定位等研究提供了基础。

SNP genotyping by High Resolution Melting Curve（ HRM） is a simple and effective,higher sensitivity and stronger specificity method. Optimization and establishment of soybean HRM- based SNP genotyping system is the precondition and basis for soybean SNP research in future. In this study,small amplicon after adding the internal calibration was used to optimize and establish SNP genotyping system. The results showed that it was better to make PCR product length 50- 80 bp,the melting temperature at 72- 82℃ and Tm value between 55- 62℃when designing a SNP primer. A total of 10μL PCR reaction system should contain 25 ng DNA template,0. 6pmol SNP primers and 1μL LC Green dye. After PCR reaction,3 pmol high and low temperature internal calibration should be added to each wells,and then denatured for HRM analysis. Moreover,the optimized SNP genotyping system was used to genotyping a recombinant inbred lines（ RILs） population,and the results demonstrated that the RILs could be completely divided into 2 genotypes of their parents by the optimized system,with improved accuracy and efficiency. The optimized SNP genotyping system established in this study will provide a useful foundation for developing SNP markers,constructing high density genetic maps,QTL identification in the future.