Isolating and confirming the MuDR-inserted flanking sequences of maize

Resumo

MuDR exhibits the highest transposition activity and insertional mutagenesis frequency in Mutator (Mu) family. If we isolate the MuDR-insertion-specific flanking sequences (MuDRFs), it will be crucial for using Mu element-mediated mutants. The MuDR-TAIL-PCR system was constructed and optimized using a combination of MuDR-TIR-nested specific primers and 12 arbitrary degenerate (AD) primers, modified reaction system and procedure and mutant DNA templates of 87 genotypes from M₂ or М_2:3 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 MuDR-TAIL-PCR, accounting for 86.60% of the total mutant-specific agarose gel bands. In addition, we confirmed the authenticity of the non-redundant flanking sequence amplifications. The amplified non-redundant flanking sequences accounted for 65.12% of the total MuDRFs, and 88.00% of the non-redundant MuDRFs were inserted inside the genes. These results show that the MuDR-TAIL-PCR system that we developed can be used for specifically isolating MuDRFs.