A competitive PCR‐based method to detect a single copy of T‐DNA insertion in transformants

Abstract

Gene function studies benefit from the availability of mutants. In plants, Agrobacterium-mediated genetic transformation is widely used to create mutants. These mutants, also called transformants, contain one or several transfer-DNA (T-DNA) copies in the host genome. Quantifying the copy number of T-DNA in transformants is beneficial to assess the number of mutated genes. Here, we developed a competitive polymerase chain reaction (PCR)-based method to detect a single copy of a T-DNA insertion in transformants. The competitor line BHK À -1 that contains a single copy of competitor BHK À (BHK, Basta, Hygromycin, Kanamycin-resistant genes) was crossed with test transformants and the genomic DNA of F 1 plants was subjected to competitive PCR. By analyzing the gray ratio between two PCR products, we were able to determine whether or not the test transformants contained a single copy of T-DNA insertion. We also generated the control lines BHK ±1:1 and BHK ±2:1 , which contain the target (BHK + ) and competitor (BHK À ) in a ratio of 1:1 and 2:1, respectively. The ratios of their PCR products are useful references for quantitative analysis. Overall, this method is reliable and simple in experimental manipulations and can be used as a substitute for Southern-blot analysis to identify a single copy of T-DNA insertion in transformants. | INTRODUCTIONIn genetics, mutants benefit the study of gene functions. Several methods, such as chemical mutagenesis, irradiation mutagenesis, and genetic transformation-mediated insertional mutagenesis, have been used to create mutants (Azpiroz-Leehan & Feldmann, 1997). In Arabidopsis thaliana and rice, Agrobacterium-mediated genetic transformation is widely used (Alonso et al., 2003;Jeon et al., 2000). This is mainly due to the establishment of an effective transformation method and the convenience of cloning the insertion sites of transfer-DNA (T-DNA). These mutants, also called transformants, contain one or several T-DNA insertions in the host genome (Tzfira et al., 2004). Integration of T-DNA into the genome usually results in gene knockout or knockdown due to transcription inactivation, incorrect splicing of primary transcripts, a shift of the translation reading frame, or unstable mRNA. It is generally believed that T-DNA integration occurs randomly in the plant genome (Azpiroz-Leehan & Feldmann, 1997), although non-random distribution of T-DNA has also been reported (Zhang et al., 2007). Theoretically, a saturated mutant library in which every gene has a corresponding T-DNA-inactivated variant can be established if the transformants are sufficient in number. Now, some methods, for example inverse PCR (Ochman et al., 1988), hiTAIL-PCR (Liu & Chen, 2007), and TADEA-PCR (Yang et al., 2018), have been developed to identify the mutated genes through cloning the insertion sites of T-DNA in the host genome, which further benefits the use of transformants in genetic studies. Hua Fan and Liu-Yuan Huang contributed equally to this work.