ABSTRACT
Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.
Subject(s)
Arabidopsis/genetics , Chromosome Mapping/methods , DNA, Bacterial/chemistry , Genome, Plant , Polymerase Chain Reaction/methods , DNA Primers , Mutagenesis, Insertional , Sequence Analysis, DNA , Transformation, GeneticABSTRACT
Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.
