Isolating and confirming the Mudr­inserted flanking sequences of maize

Abstract

MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDR­insertion­specific flanking sequences (MuDRFs), it will be crucial for using Mu element­mediated mutants. The MuDR­TAIL­PCR system was constructed and optimized using a combination of MuDR­TIR­nested specific primers and 12 arbitrary degenerate (AD) primers, modified reaction system and procedure and mutant DNA templates of 87 genotypes from M2 or M2:3 families created by crossing the W22::Mu line (active MuDR donor parent) from the UniformMu population with the Zong31 (Z31) line (recipient parent). Here 129 different MuDRFs were acquired by MuDR­TAIL­PCR, accounting for 86.60 % of the total mutant­specific agarose gel bands. In addition, we confirmed the authenticity of the non­redundant flanking sequence amplifications. The amplified non­redundant flanking sequences accounted for 65.12 % of the total MuDRFs, and 88.00 % of the non­redundant MuDRFs were inserted inside the genes. These results show that the MuDR­TAIL­PCR system that we developed can be used for specifically isolating MuDRFs.