Application of competitive PCR for screening selectable marker-free Xa21 transgenic rice

Abstract:

Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa21 transgenic rice plants. The competitive template of Xa21 was the endogenous Xa21 homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.