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

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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.

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Acknowledgements

We are grateful to Dr. Guo-liang Wang for critical reading of the manuscript. The work was supported by The Chinese Academy of Sciences (grant No. KXCX2-1-02-01), and The Basic Research Program (The '973' Program, grant No. TG2000016203) and High-Tech Program (The '863' Program, grant No. 2001AA225012) of the Ministry of Science and Technology of China.

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  1. Laboratory of Plant Biotechnology, Institute of Microbiology, The Chinese Academy of Sciences, Zhong Guan Cun, 100080, Beijing, P.R. China

    Y. Sha, S. Li, Z. Pei, L. Luo, Y. Tian & C. He

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  1. Y. Sha
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  2. S. Li
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Correspondence to C. He.

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Communicated by D. Mackill

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Sha, Y., Li, S., Pei, Z. et al. Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 108, 306–314 (2004). https://doi.org/10.1007/s00122-003-1423-9

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