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 M23 families created by crossing the W22Mu 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.