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为提高基于InDel-Pyrosequencing的黄瓜杂交种纯度检测通量,降低检测成本,本研究模拟10种DNA Pooling进行PCR、Pyrosequencing及等位基因频率分析。通过TTest分析不同Pooling间等位基因频率的差异性,确定3 Pooling为种子纯度检测最适Pooling数;建立3 Pooling-InDel-Pyrosequencing标准曲线,其R2达0.999 1;根据该标准曲线,检测黄瓜杂交品种“园中王”30粒杂交种种子纯度,结果为96.67%。本研究丰富了基于InDel-Pyrosequencing的黄瓜杂交种纯度检测技术体系。
In order to increase the purity of the cucumber hybrids based on InDel-Pyrosequencing, and to reduce the cost of detection, ten DNA pooling assays were performed for PCR, Pyrosequencing and allele frequency analysis. According to the standard curve, the hybridization of cucumber was detected by using the three-pooling method, and the difference of allele frequency among different Pooling was analyzed by TTest. 3 Pooling was the optimum Pooling number for seed purity detection; 3 Pooling-InDel-Pyrosequencing standard curve was established with R2 of 0.999 1; Variety “garden king ” 30 hybrid seed purity, the result was 96.67%. This study enriched the purity detection system of cucumber hybrids based on InDel-Pyrosequencing.