应用高分辨率熔解曲线技术分析水稻分子标记基因型

 【目的】验证高分辨熔解曲线（high-resolution melting curve analysis，HRM）技术在水稻分子标记分析中应用的可行性和可靠性，应用该技术对水稻重组自交系（RIL）群体及其亲本进行SSR、InDel和SNP标记基因型分析。【方法】以粳稻品种丽江新团黑谷（LTH）和籼稻品种三黄占2号（SHZ-2）及其衍生的RIL群体为材料，以纯合亲本LTH作为参比基因型，利用HRM技术分析一个G/A转换的SNP标记以及非变性聚丙烯酰胺凝胶电泳（PAGE）难以分辨的一个SSR标记和一个InDel标记的基因型。【结果】通过加入适量的参比DNA，HRM能够分辨2个纯合亲本及其衍生的纯合体和杂合体RI系的分子标记基因型，并且以加入20%模板量的参比DNA对3种基因型的分辨最好。利用该方法对作图群体的部分RI系进行分子标记基因型分析，结果与变性PAGE的分析结果完全吻合。【结论】验证了HRM应用于水稻SSR、InDel以及SNP标记分析的可行性和可靠性。相对于凝胶电泳分析，HRM具有分辨率高、高效、快速、操作简单、安全等优点，是一种在水稻分子标记分析中很有应用前景的技术。

Application of High-Resolution Melting Curve Analysis for Molecular Marker Genotyping in Rice

【Objective】 In order to test the feasibility and reliability of high-resolution melting curve analysis (HRM) for molecular marker genotyping in rice crop, SSR, InDel and SNP genotyping of a rice recombinant inbred line (RIL) population and their parents were conducted using HRM. 【Method】 A japonica cultivar Lijiangxintuanheigu (LTH), an indica cultivar Sanhuangzhan 2 (SHZ-2) and their derived RIL populations were used. The amplicons of the two molecular markers, a SSR marker and an InDel marker, which could not be differentiated by non-denaturing polyacrylamide gels electrophoresis (PAGE), and the amplicons of a G/A transition SNP marker, were subjected to HRM analysis. The reference homozygous DNA from LTH was added before PCR. 【Result】 By adding reference DNA, the heterozygous and two different homozygous genotypes generated from SSR, InDel and SNP, respectively, could be distinguished by HRM. The best discrimination for the three genotypes was given when the amount of reference DNA was equal to 20% template DNA. The genotypes of two parents and their derived RI lines based on HRM were perfectly consistent with those based on denaturing PAGE. 【Conclusion】 HRM can be used for SSR, InDel and SNP genotyping in rice. Comparing with PAGE, HRM has the advantages of high resolution, high through-put, simplicity and safety. It is a promising technology for molecular marker analysis in rice.