3D-PCR Screening System of Retrotransposon Tos17 Insertion Lines with Next Generation Sequencing

Endogenous retrotransposon Tos17 in rice is only active in cultured cells. We have generated about 50,000 Tos17-insertion lines by regeneration of cultured rice cells. Ten copies of Tos17 in average were found to be transposed in a given regenerated rice line.　We have already constructed three dimensional DNA pools for PCR screening using primers which have an end-sequence of Tos17 and sequence of target gene. However, the screening method requires a lot of PCR reactions. To reduce steps, we devised a new procedure: Using DNA pools for first screening, flanking sequences were determined by the next-generation sequencing. Each DNA of pool was fragmented by sonication. After blunting of ends of fragments, fragments were ligated with adapter. Flanking regions from each DNA pool were amplified by suppression PCR method using primers for the Tos17-end sequence and the adapter sequence which have unique sequence tags (5 base length). The 49 sets of amplified product were mixed and then sequenced by HiSeq2000. Sequences which have primer sequences for Tos17 or adapter were selected and then grouped by the 5-base unique tag. Junctions of Tos17 were identified by BLAST search. Approximately 8000 inserted loci of Tos17 on the reference genome were detected. Using the inserted position of the target gene and using the information of DNA pool identified with the tag, Tos17 insertion mutant will be identified by the second PCR screening. This system will be helpful for screening of insertion mutants, which leads to functional analysis of rice genes.