基于KASP平台的转基因玉米高通量特异性检测方法

High-throughput Specific Detection Methods for Transgenic Maize Based on the KASP Platform

为了促进转基因玉米产业化发展,加快优良性状转化体与骨干自交系的转育工作,适用于回交群体的高通量前景选择方法亟待开发。本研究以转基因玉米DBN9936为材料,根据其外源插入片段及其侧翼序列,采用primer5软件设计了6对特异性引物,与内标准基因zSSIIb引物进行组合用于评估,获得最优引物组合后,进一步开展特异性验证、检出限测试和多样品验证。结果显示,最优引物组合为DBN9936-LB1*zSSIIb-k1;10份DBN9936 BC1代种子的KASP基因分型结果与琼脂糖凝胶电泳结果完全一致;10份转化体DBN9858、DBN9501、C0010.1.3、C0010.3.1、C0010.3.7、C0030.2.5、BT11、GA21、MIR162和2A-7的转基因玉米样品在双平台上检测结果均表现为阴性;转基因玉米DBN9936的检出限为10%;48份不同DBN9936杂交种材料的KASP基因分型结果均表现为阳性。综上,本研究方法可用于转基因玉米DBN9936回交转育过程中的前景选择,且样品通量高,为其他农作物在转基因育种过程中的目标基因检测提供了技术参考。

In order to promote the industrialization of transgenic maize and accelerate the transfer of excellent trait transformants and backbone inbred lines,high-throughput foreground selection methods for backcross population need to be developed urgently.In this study,the transgenic maize DBN9936 was used as the material,according to its exogenous insert fragment and its flanking sequence,6 pairs of specific primers were designed using the primer5 software,combined with the internal standard gene zSSIIb primer for evaluation.After obtaining the optimal primer combination,further the specificity verification,detection limit testing and multi-sample verification were carried out.The results showed that the optimal primer combination was DBN9936-LB1*zSSIIb-k1;the results of KASP genotyping of 10 DBN9936 BC1 seeds were completely consistent with those of agarose gel electrophoresis;the results of 10 transformants DBN9858,DBN9501,C0010.1.3,C0010.3.1,C0010.3.7,C0030.2.5,BT11,GA21,MIR162 and 2A-7 were negative on the dual platform.The detection limit of transgenic maize DBN9936 was stable at 10%;and the KASP genotyping results of 48 different DBN9936 hybrids were positive.In summary,this method can be used for the foreground selection in the process of backcrossing of transgenic maize DBN9936 with high sample throughput,which provides a technical reference for target gene detection of other crops in transgenic breeding.