水稻SSR-SNP组合型分子标记的开发与应用

As a classical genetic marker, SSR has higher polymorphism than SNP but its accuracy of genotyping is lower than that of SNP. In the previous study, we achieved high-throughput SSR genotyping at single-base resolution for the first time and found a large number of genomic regions with both SNP and SSR simultaneously, which could be used as combinatorial molecular markers and have the potential to be more polymorphic and more accurate. Firstly, this project aims to develop a new method used for achieving accurate reads mapping and SNP calling, so the high-throughput SSR genotyping method established in our early study could be improved and used to realize the precise genotyping of the combinatorial molecular markers. The distribution and polymorphism of SNPs around SSRs will be analyzed at the whole genome level and 600 candidate combinatorial SSR-SNP molecular markers with high polymorphism and even distributed in the genome will be selected. 100 rice varieties will be selected to further estimate the amplification efficiency and polymorphism of these markers, and 500 combinatorial markers with best performance will be retained. Based on the 500 markers, a DNA fingerprint databases of 600 authorized rice varieties could be established and the advantage of high polymorphic, accurate and discrimination of the new markers could be verified. The novel combinatorial molecular markers and the genotyping method developed in this project have the advantages of high polymorphism and high accuracy, and can be widely used in rice genetic analysis.

作为经典的遗传标记，SSR的多态性高于SNP但分型准确性低于SNP。前期研究中，我们首次实现了高通量、单碱基分辨率的SSR分型，并发现大量同时具有SNP和SSR的组合型标记区域，该区域具多态性更高、分型更准确的潜力。本项目拟针对SSR区域序列不易准确比对的问题开发一套新的SNP分析方法，以此改进前期建立的高通量的SSR分型方法，实现组合型标记的精准分型；利用水稻基因组重测序数据在全基因组水平分析组合型标记区域中SNP的分布规律和多态性，选取600个多态性高且在基因组上均匀分布的候选组合型标记；选取100个水稻品种进一步分析这些标记的扩增效率和多态性，保留500个综合性能最佳的SSR-SNP组合型标记；基于上述标记建立600个已授权品种的DNA指纹数据库，并验证新型标记在多态性、准确性和品种区分度上的优势。本项目开发的新型标记及其检测技术具有高多态性、高准确性特点，可广泛用于水稻遗传学分析。

SSR是目前广泛使用的分子标记之一，但其分型准确性容易收到DNA聚合酶的滑动干扰，影响分型准确性。本项目在全基因组水平筛选同时具有SSR和SNP的基因组区域作为新型分子标记，利用SSR标记中的SNP信息提高分型准确。我们利用4000多份水稻基因组重测序数据分析了全基因组水平的变异信息和SSR分布，从中筛选了一批具有高多态性的分子标记位点，并成功设计了多重PCR引物。针对新型分子标记的特点开发了一套完善的分型方法，重现性达到99.80%。此外，我们还开发一种称为MNP的分子标记，并把这批分子标记列入了植物品种鉴定的国家标准。同时，我们还完成了历年收集的607个水稻样品的测序和分型，结果显示常规稻样品平均每个样品有1.21%的位点为杂合位点，最高为9.89%；杂交稻中有25%以上的位点为杂合位点。这表明常规稻中也会存在杂合位点，这不利于保持水稻品种的遗传稳定性，而稳定性是品种授权的基本要求之一。所有样品两两比较时平均有41.75%的位点分型结果有差异，最大为76.71%；仅有0.09%的样品对差异位点比例小于4%。同时，粳稻和籼稻品种间差异明显，聚类分析中也分为两个完全不同簇。按年份对所有品种分析后发现，随着品种数量逐年增加，新出现品种贡献的特异等位基因型数量在逐年递减，表明育种过程中并未持续大量引入新的遗传资源。