Cloning Flanking Sequences And Genetic Analysis Of Leaf Color Mutant Of Maize Inserted By Mutator Transposon

Maize genome sequencing for corn functional genomics research opened a new chapter, also for maize germplasm creation and molecular breeding has laid an important foundation. Mutants are the most ideal and the most effective material for reverse genetics research to isolation and clone gene, and analysis gene functional. Mutator (Mu) transposon is maize endogenous transposon, having high mutation characteristics, extremely easy to obtain diversity genetic of the mutant. At the same time the transposon tagging technique is a important strategy to develop maize functional genomics research. This study with active MuDR transposon W22:: Mu as male parent, hybrid with elite inbred lines Z31. After many generations of selfing or backcross pollination, screening out Mu transposon mutagenesis of leaf color mutants, as this study of Mu transposon tagging technique for separating the leaf color mutant target genetic material. The use of modified MuTail-PCR, MuAFLP and Illumina sequencing methods, separation of Mu insertion flanking sequences, and the Mu insertion site gene carried by bioinformatics analysis and verification of authenticity, obtain the result:1, Cloning 129 flanking sequences with Mu-TIR; in which 36 sequence are repeats sequence, and 93 specific sequences. 18 sequences of the specific sequences of insertion sites are multiple copy sequences, accounted for 18.9%. The specificity of single-copy sequences of the remaining 75 sequences on maize chromosomes of the distribution location: on the 4, 6 and 7 chromosome distribution less, are respectively 2.67%,1.33% and 4%, for a total of 8%; the 2, 5, 8 and 9 chromosome more distributed, are respectively 13.33%, 13.33%, 16% and 18.67%, for a total of 61.33%. The 75 sequences average length is about 600bp, which in 200 ~ 400bp range has 6 sequences; in 400 ~ 600bp range has 26 sequences;, in 600 ~ 1000bp range with 43sequencea, and no less than 200bp sequence.2,To analysis the 9 bp forward repeat sequences on 75 sequences, discovered 5 Mu factor insertion hotspot sequence: GTCACGCAC, GTTAGCAGA, CHTTYCTAG, ATTCTCTAK, TGCAGTACA.3, Using MuAFLP method to obtain different memory expansion from leaf color mutant pedigrees, with recovery and sequencing, the sequencing results removal of the carrier and Mu-TIR sequences, obtain a total of 14 Mu insertion site of the target sequence. Removal 2 redundant sequenceand and 8 repeat sequences, only 4 target sequences as a further the object of study. And 2 sequences as the authenticity of the insertion sites.4, PPR mutation site positioned on the 6 chromosome of maize, and Mu factor insertion in the gene 5’ UTR District, located at 121st base and 122nd base; this mutation segregating population genotype proportions with Mendel separation rule, but not associated with phenotype. 5, PB1 mutation positioned on 5 chromosome of maize, Mu factor insertion in exon 1 of the gene. This mutation segregating population genotype proportions with Mendel separation rule, but with the phenotype without comorbid separation.