Researching The Methods Of Isolating The Flanking Sequences Of Maize Mutants Inserted By MuDR Transposon

Maize is not only the most planted grain crop, but also the major model plant species for genetics and transposons research. The functional genomic study is more and more deeply after the sequencing job and functional genomic study is becoming the major aspect of genome research. Mutants are good resourses for genetic reserches. Mutators(Mu) are the endogenous transposons and they are the most effectious tools for creating defect mutants. Thus, transposon tagging technology is the important basis and foundations for study the flanking sequences inserted by transposons.Elite maize inbred line Zong31(Z31, female parent) and Mu DR activity parent W22::Mu(male parent) were crossed to construct the mutagenized populations. We acquired maize kernel fullness mutants and viviparous mutants and the mutants were used to optimize and improve the Mu DR-AFLP methods. At the same time, the the mutants were used for phenotype evaluation, genetic analysis and cloning the insertion flanking sequences to associating them with the Mu DR insertions. The major results were as follows:1. W22::Mu and Z31 were used to construct M2 mutagenized populations. After genetic analysis, six maize kernel fullness mutants and one viviparous mutant were acquired, and they were all used for constructing the Mu DR-AFLP system. Z31 were used as recurrent parent to cross with the site-containing materials to construct BC4F2:3 populations. The genetic nanalysis of the segregated populations showed us that, the seven mutants were all controlled by few nuclear genes. At the same time, we analized the volume-weight of maize kernel fullness mutatnts: the volume-weight of hollow kernels were significantly diffrent with their corresponding wild kernels(P<0.05).2. M2:3 mutant families were used as the materials, Sac I enzyme site was designed at Mu DR-TIR specific region; Mse I enzyme site was designed at genomic sequence to construct the best enzyme combination. The enzyme combination was used to digest the Mu DR insertions mutants’ genome, and we constructed the Mu DR-AFLP system to isolate the mutant specific flanking sequences with Mu DR insertion.3. Six Mu DR inserted flanking sequences were isolated at last using our constructed Mu DR-AFLP system, and the authenticity of Mu DR insertion were verified. In another word, they were truly inserted by Mu DR post-transpoition. In the populations, we found one flanks co-segregated with the mutant phenotype—maize kernel fullness phenotype.4. Based on the bioinformatics analysis of the Mu DR insertion flanks, genetic analysis of mutant traits and the mutant phenotype valuations, we constructed the association between mutant traits and Mu DR insertions, created precious materials for the further gene function parsing.