CAJ | 학술논문

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；10μL PCR反应体系中应包含25ng DNA模板，0.6pmol SNP引物，1μL LC Green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。
응용고분변솔용해곡선（ high resolution melting curve，HRM）기술진행기인분형간단유효，령민도고、특이성강。우화병건립기우HRM적대두SNP기인분형체계，시준학진행SNP연구적전제화기출。본연구이용첨가내표후적소편단법진행SNP기인분형체계우화연구，결과표명재설계SNP인물시，최호사기PCR산물장도위50~80bp，용해온도재72~82℃，Tm치개우55~62℃지간；10μL PCR반응체계중응포함25ng DNA모판，0.6pmol SNP인물，1μL LC Green염료；PCR반응후，응재각점양공분별가입3pmol적고、저온내표，연후진행변성처리화HRM분석。동시이용건립적기인분형체계대중조자교계군체（ RIL）진행료기인분형검측，능완전장기분성친본적량충기인형，제고료SNP기인분형적준학성화효솔。본연구건립적SNP기인분형체계위금후진행대두SNP표기개발、고밀도유전도보구건、QTL정위등연구제공료기출。
SNP genotyping by High Resolution Melting Curve( HRM)is a simple and effective,higher sensi-tivity and stronger specificity method. Optimization and establishment of soybean HRM-based SNP genotyping sys-tem 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 -80bp,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 25ng 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.