Generation and Analysis of End Sequence Database for T-DNA Tagging Lines in Rice1

We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.

[1]  Richard G. F. Visser,et al.  Fluorescencein situhybridization on extended DNA fibres as a tool to analyse complex T‐DNA loci in potato , 1998 .

[2]  S. Yamamoto,et al.  The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets , 2001, Molecular Genetics and Genomics.

[3]  M. Cho,et al.  Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. , 1999, The Plant journal : for cell and molecular biology.

[4]  Rossana Henriques,et al.  Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes. , 2002, The Plant journal : for cell and molecular biology.

[5]  T. Komari,et al.  Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. , 1994, The Plant journal : for cell and molecular biology.

[6]  H. Hirochika,et al.  Applications of retrotransposons as genetic tools in plant biology. , 2001, Trends in Plant Science.

[7]  K. Feldmann,et al.  T-DNA insertion mutagenesis in Arabidopsis: going back and forth. , 1997, Trends in genetics : TIG.

[8]  B. Reiss,et al.  Isolation of a gene encoding a novel chloroplast protein by T‐DNA tagging in Arabidopsis thaliana. , 1990, The EMBO journal.

[9]  S. Dellaporta,et al.  Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis , 2000, Functional & Integrative Genomics.

[10]  P. Springer.  Gene Traps: Tools for Plant Development and Genomics , 2000, Plant Cell.

[11]  V. Walbot,et al.  Saturation mutagenesis using maize transposons. , 2000, Current opinion in plant biology.

[12]  W. Stiekema,et al.  A Two-Component Enhancer-Inhibitor Transposon Mutagenesis System for Functional Analysis of the Arabidopsis Genome , 1999, Plant Cell.

[13]  H. Hirochika,et al.  Retrotransposons of rice involved in mutations induced by tissue culture. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. An,et al.  T-DNA Insertional Mutagenesis for Activation Tagging in Rice1 , 2002, Plant Physiology.

[15]  Jonathan D. G. Jones,et al.  Function Search in a Large Transcription Factor Gene Family in Arabidopsis: Assessing the Potential of Reverse Genetics to Identify Insertional Mutations in R2R3 MYB Genes , 1999, Plant Cell.

[16]  A. Oliphant,et al.  A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). , 2002, Science.

[17]  Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA. , 1999, BioTechniques.

[18]  Cari Soderlund,et al.  In-Depth View of Structure, Activity, and Evolution of Rice Chromosome 10 , 2003, Science.

[19]  N. Fedoroff,et al.  A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana , 2002, Plant Molecular Biology.

[20]  S. Goff,et al.  A High-Throughput Arabidopsis Reverse Genetics System Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004630. , 2002, The Plant Cell Online.

[21]  D. Bouchez,et al.  Arabidopsis gene knockout: phenotypes wanted. , 2001, Current opinion in plant biology.

[22]  H. Hirochika.  Contribution of the Tos17 retrotransposon to rice functional genomics. , 2001, Current opinion in plant biology.

[23]  H. Saedler,et al.  Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Ronald,et al.  A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications , 2004, Plant Molecular Biology Reporter.

[25]  Jonathan D. G. Jones,et al.  ATIDB: Arabidopsis thaliana insertion database. , 2003, Nucleic acids research.

[26]  Jonathan D. G. Jones,et al.  Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. , 1995, Genes & development.

[27]  C. Koncz,et al.  T-DNA insertional mutagenesis in Arabidopsis , 1992, Plant Molecular Biology.

[28]  H. Saedler,et al.  Technical advance: display and isolation of transposon-flanking sequences starting from genomic DNA or RNA. , 2000, The Plant journal : for cell and molecular biology.

[29]  Kazuo Shinozaki,et al.  A New Resource of Locally Transposed DissociationElements for Screening Gene-Knockout Lines in Silico on the Arabidopsis Genome1,212 , 2002, Plant Physiology.

[30]  M. Sussman,et al.  T-DNA as an Insertional Mutagen in Arabidopsis , 1999, Plant Cell.

[31]  Yujun Zhang,et al.  Sequence and analysis of rice chromosome 4 , 2002, Nature.

[32]  E. Ábrahám,et al.  Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. , 2002, The Plant journal : for cell and molecular biology.

[33]  Kazuhiro Kikuchi,et al.  The plant MITE mPing is mobilized in anther culture , 2003, Nature.

[34]  C. Koncz,et al.  Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension. , 1998, The Plant journal : for cell and molecular biology.

[35]  M. Van Montagu,et al.  T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation , 2000, Molecular Breeding.

[36]  Jonathan D. G. Jones,et al.  Multiple Independent Defective Suppressor-mutator Transposon Insertions in Arabidopsis: A Tool for Functional Genomics , 1999, Plant Cell.

[37]  M. Fladung,et al.  Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome , 2000, Molecular and General Genetics MGG.

[38]  V. Sundaresan,et al.  Analysis of Flanking Sequences from Dissociation Insertion Lines: A Database for Reverse Genetics in Arabidopsis , 1999, Plant Cell.

[39]  K. Jung,et al.  T-DNA insertional mutagenesis for functional genomics in rice. , 2000, The Plant journal : for cell and molecular biology.

[40]  K. Feldmann.  T‐DNA insertion mutagenesis in Arabidopsis: mutational spectrum , 1991 .

[41]  Hong-Gyu Kang,et al.  Transgene structures in T-DNA-inserted rice plants , 2003, Plant Molecular Biology.

[42]  G. Pelletier,et al.  In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. , 1998, Methods in molecular biology.

[43]  T. Gojobori,et al.  The genome sequence and structure of rice chromosome 1 , 2002, Nature.

[44]  L. Križková,et al.  Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. , 1998, The Plant journal : for cell and molecular biology.

[45]  K. Shimamoto,et al.  Ac as a tool for the functional genomics of rice. , 1999, The Plant journal : for cell and molecular biology.

[46]  M. Schmid,et al.  Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana , 2003, Science.

[47]  Huanming Yang,et al.  A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica) , 2002, Science.

[48]  Xianghe Yan,et al.  Use of the Transposon Ac as a Gene-Searching Engine in the Maize Genome , 2002, The Plant Cell Online.

[49]  S. Gelvin,et al.  Integration of T-DNA binary vector 'backbone' sequences into the tobacco genome: evidence for multiple complex patterns of integration. , 1997, The Plant journal : for cell and molecular biology.

[50]  P. Abad,et al.  Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study. , 2002, Comptes rendus biologies.

[51]  E. Hui,et al.  Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants , 1998, Cellular and Molecular Life Sciences CMLS.

[52]  Yaoguang Liu,et al.  Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. , 1995, Genomics.

[53]  V. Sundaresan,et al.  Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project. , 2000, Current opinion in biotechnology.