摘要
植物中广泛分布着单核苷酸多态性(SNP)位点。在此基础上发展而来的SNP标记因其具有高分辨率和共显性等优点,已成为当前作物遗传研究重要的分子工具。本研究拟建立基于高分辨率熔解曲线(HRM)技术的SNP分子标记,从而实现对栽培稻和野生稻的高效基因分型,为今后水稻的基因挖掘、品种鉴定以及分子育种等提供可靠、快捷的技术工具。利用水稻全基因组9 K SNP芯片对栽培稻品种黄华占和野生稻Y605进行扫描,寻找两者之间的SNP位点,并将其开发成基于HRM技术的特异分子标记。然后,利用这些分子标记对亲本黄华占、野生稻Y605以及两者的BC3回交群体进行分子检测,以验证其有效性。水稻9 K基因芯片在黄华占与野生稻Y605之间总共找到了4198个SNP位点,它们在12条染色体上较均匀分布。在水稻第1号染色体上随机挑选出5个SNP位点开发成基于HRM技术的特异分子标记。利用这些标记对黄华占与野生稻Y605的BC3F1和BC3F2群体进行检测分析,发现它们都能准确区分亲本的纯合与杂合基因型。并且,在回交后代的第1号染色体ZY1-1~ZY1-4标记区间检测到野生稻片段插入。水稻全基因组9 K SNP芯片可以很好地应用于水稻SNP标记的开发。开发的SNP特异标记能准确、高效地对栽培稻和野生稻进行基因分型。进一步完成基于HRM技术的水稻全基因组SNP标记的开发,可为今后野生稻的分子遗传研究、有利基因挖掘和育种应用提供高效的分子检测手段。
Single nucleotide polymorphism(SNP) sites are widely distributed in plants. SNPs-based markers have become an important molecular tool for crop genetic research due to their high resolution and co-dominant. This study aims to develop SNP molecular markers based on high resolution melting(HRM) technology and to estimate their genotyping efficiency between cultivated rice and wild rice,thus providing a reliable,simple and rapid tool for gene discovery,variety identification and molecular breeding in the future. Genome-wide scanning of SNPs was performed between the cultivar Huanghuazhan and wild rice Oryza rufipogon Griff. Y605 using the rice 9 K SNP microarray. Then we selected and developed HRM technology-based molecular markers from these SNPs. These markers were subsequently used for genotyping of BC3 backcrossed populations with their parents Huanghuazhan andY605 to verify their validity. A total of 4198 SNPs were found,almost evenly distributed on all the chromosomes.Five randomly selected SNPs from the first chromosome were employed for conversion into HRM technology-based markers. These markers were accurate and efficient in genotyping of BC3 F1 and BC3 F2 populations of Huanghuazhan and wild rice Y605,as well as the homozygous parents and F1 heterozygous. In addition,the introgression fragments from wild rice were detected in the ZY1-1-ZY1-4 marker interval of the first chromosome of the backcross. The genome-wide rice 9 K SNP microarray can be well applied to explore the polymorphism sites for developing user-friendly SNP markers,which are accurate and efficient in use of genotyping cultivated rice and wild rice. Further development of genome-wide rice SNP markers based on HRM technology will provide an efficient molecular detection tool for molecular genetic research,favorable genes discovery and breeding applications of wild rice.
引文
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