Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize

摘要: High-copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype-driven forward genetics has hampered by the difficulty identifying specific insertions responsible phenotypes interest. We describe new method that can substantially increase throughput linking disrupted gene to known phenotype high-copy Mutator (Mu) transposon lines maize. The approach uses Illumina platform obtain sequences flanking Mu elements pooled, bar-coded DNA samples. Insertion sites are compared among individuals suitable genotype identify those linked mutation is prepared sequencing mechanical shearing, adapter ligation, and selection fragments harboring hybridization biotinylated oligonucleotide corresponding terminal inverted repeat. This yields dense clusters sequence reads tile approximately 400 bp each side heritable insertion. demonstrated causal four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), expression (polynucleotide phosphorylase PTAC12), prosthetic group attachment (HCF208/CCB2). adds tools available tagging maize, could be adapted use with other transposons. A by-product identification numerous unrelated targeted phenotype, which contribute community insertion resources.