应用高分辨率熔解曲线技术分析水稻分子标记基因型

doi:10.3864/j.issn.0578-1752.2011.18.001

中国农业科学 ›› 2011, Vol. 44 ›› Issue (18): 3701-3708.doi: 10.3864/j.issn.0578-1752.2011.18.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

应用高分辨率熔解曲线技术分析水稻分子标记基因型

赵均良, 张少红, 刘斌

1. 广东省农业科学院水稻研究所

收稿日期:2011-01-12 修回日期:2011-03-08 出版日期:2011-09-15 发布日期:2011-09-15
通讯作者: 通信作者刘斌，Tel：020-85517279；E-mail：lbgz_2006@tom.com
作者简介:赵均良，Tel：020-87571952；E-mail：otest@163.com。张少红，Tel：020-87571952；E-mail：szhanggz@tom.com。张少红与赵均良为同等贡献作者
基金资助:
国家自然科学基金项目（30671251，30771392，30821140350）、广东省自然科学基金（重点）项目（07117962，9251064001000001）、广东省农业科学院重点基础研究项目（07-基础-04）

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

ZHAO Jun-Liang, ZHANG Shao-Hong, LIU Bin

1. 广东省农业科学院水稻研究所

Received:2011-01-12 Revised:2011-03-08 Online:2011-09-15 Published:2011-09-15

摘要/Abstract

摘要： 【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。

关键词:

水稻, SSR标记, InDel标记, SNP标记, 高分辨率熔解曲线

Abstract: 【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.

Key words: rice, SSR, Indel, SNP, HRM

赵均良, 张少红, 刘斌. 应用高分辨率熔解曲线技术分析水稻分子标记基因型[J]. 中国农业科学, 2011, 44(18): 3701-3708.

ZHAO Jun-Liang, ZHANG Shao-Hong, LIU Bin. Application of High-Resolution Melting Curve Analysis for Molecular Marker Genotyping in Rice[J]. Scientia Agricultura Sinica, 2011, 44(18): 3701-3708.

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应用高分辨率熔解曲线技术分析水稻分子标记基因型

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

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