Generation and flanking sequence analysis of a rice T-DNA tagged population.

Abstract

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice ( Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T1 generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.