A direct PCR-based SNP marker-assisted selection system (D-MAS) for different crops
A direct PCR-based SNP marker-assisted selection system (D-MAS) for different crops
复制标题
针对不同作物的基于直接 PCR 的 SNP 标记辅助选择系统 (D-MAS)
DOI：
10.1007/s11032-019-1091-3
复制
发表时间：
2020-01-04
影响因子：
3.1
通讯作者：
Li, Xu
中科院分区：
文献类型：
--
作者：
Lu, Jun;Hou, Jing;Li, Xu
随着越来越多的作物基因组重测序数据的公布，单核苷酸多态性（single nucleotide polymorphisms，SNPs）是最容易获得的基因分型标记，在各种分子标记中表现出最高的丰度。然而，高通量的SNP基因分型方法，如KASP（竞争等位基因特异性PCR）、TaqMan或ARMS（扩增难治突变系统），通常是DNA提取的劳动，并且依赖于昂贵的设备。因此，大多数育种家都面临着缺乏一个简单，节省成本和稳定的基因分型系统的瓶颈。在这里，我们报告了一个直接基于PCR的中等通量SNP标记辅助选择（D-MAS）系统适用于现有的主要作物，包括水稻，小麦，玉米和油菜籽。D-MAS系统包括（1）温室育苗;（2）碱裂解法高通量DNA提取;（3）PARMS（penta-primer amplification refractory mutation）或KASP基因分型系统384孔无凝胶SNP标记检测。通过灵活的稀释倍数和较长的低温保存时间验证了碱裂解DNA的稳定性。水稻、小麦、玉米和油菜四个F2群体的碱裂解DNA与十六烷基三甲基溴化铵（CTAB）法相比，在SNP识别上表现出相同的效率。在碱裂解DNA的情况下，PAMRS比KASP显示出更密集的基因型簇。此外，D-MAS系统对水稻老叶具有较强的适应性。在杂交水稻种子纯度检测中，利用籼粳特异的SNP标记验证了D-MAS系统的通量和效率，该标记也可用于水稻分子育种中1 - 2d基因的筛选。综上所述，我们提出了一个直接基于PCR的SNP调用管道，这可能是一个简单，廉价，和强大的标准操作程序（SOP）的分子育种不同的作物，并得到广泛的使用，在大多数实验室。
As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to selectipa1-2dgene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.