Plant Molecular Biology

, Volume 65, Issue 5, pp 587–601 | Cite as

Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library

  • Pietro Piffanelli
  • Gaétan Droc
  • Delphine Mieulet
  • Nadège Lanau
  • Martine Bès
  • Emmanuelle Bourgeois
  • Claire Rouvière
  • Fréderick Gavory
  • Corinne Cruaud
  • Alain Ghesquière
  • Emmanuel GuiderdoniEmail author


We characterized the insertion sites of newly transposed copies of the tissue-culture-induced ty1-copia retrotransposon Tos17 in the Oryza Tag Line (OTL) T-DNA mutant library of rice cv. Nipponbare. While Nipponbare contains two native copies of Tos17 the number of additional copies, deduced from Southern blot analyses in a subset of 384 T-DNA lines and using a reverse transcriptase probe specific to the element, ranged from 1 to 8 and averaged 3.37. These copies were shown to be stably inherited and to segregate independently in the progenies of insertion lines. We took advantage of the absence of EcoRV restriction sites in the immediate vicinity of the 3′ LTR of the native copies of Tos17 in the genome sequence of cv. Nipponbare, thereby preventing amplification of corresponding PCR fragments, to efficiently and selectively amplify and sequence flanking regions of newly transposed Tos17 inserts. From 25,286 T-DNA plants, we recovered 19,252 PCR products (76.1%), which were sequenced yielding 14,513 FSTs anchored on the rice pseudomolecules. Following elimination of redundant sequences due to the presence of T-DNA plants deriving from the same cell lineage, these FSTs corresponded to 11,689 unique insertion sites. These unique insertions exhibited higher densities in subtelomeric regions of the chromosomes and hot spots for integration, following a distribution that remarkably paralleled that of Tos17 sites in the National Institute for Agrobiological Sciences (NIAS) library. The insertion sites were mostly found in genic regions (77.5%) and preferably in coding sequences (68.8%) compared to unique T-DNA insertion sites in the same materials (49.1% and 28.3%, respectively). Predicted non- transposable element (TE) genes prone to a high frequency of Tos17 integration (i.e. from 5 to 121 inserts) in the OTL T-DNA collection were generally found to be also hot spots for integration in the NIAS library. The 9,060 Tos17 inserts inserted into non TE genes were found to disrupt a total of 2,773 genes with an average of 3.27 inserts per gene, similar to that in the NIAS library (3.28 inserts per gene on average) whereas the 4,472 T-DNA inserted into genes in the same materials disrupted a total of 3,911 genes (1.14 inserts per gene on average). Interestingly, genes disrupted by both Tos17 and T-DNA inserts in the library represented only 14.9% and 10.6% of the complement of genes interrupted by Tos17 and T-DNA inserts respectively while 52.1% of the genes tagged by Tos17 inserts in the OTL library were found to be tagged also in the NIAS Tos17 library. We concluded that the first advantage in characterizing Tos17 inserts in a rice T-DNA collection lies in a complementary tagging of novel genes and secondarily in finding other alleles in a same genetic background, thereby greatly enhancing the library genome coverage and its overall value for implementing forward and reverse genetics strategies.


Insertion mutagenesis Rice T-DNA Tos17 



We wish to thank Jean benoît Morel for his help in MPSS analysis. This project was supported by the Génoplante programme (M1 project) and benefited from a granted project at the French National Sequencing Centre (Génoscope). This work was carried out under the infrastructures of the Génopole Montpellier Languedoc Roussillon.

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  1. An SY, Park S, Jeong DH, Lee DY, Kang HG, Yu JH, Hur J, Kim SR, Kim YH, Lee M, Han SK, Kim SJ, Yang JW, Kim E, Wi SJ, Chung HS, Hong JP, Choe V, Lee HK, Choi JH, Nam JM, Kim SR, Park PB, Park KY, Kim WT, Choe S, Lee CB, An GH (2003) Generation and analysis of end sequence database for T-DNA tagging lines in rice. Plant Physiol 133:2040–2047CrossRefPubMedGoogle Scholar
  2. Balzergue S, Dubreucq B, Chauvin S, Le-Clainche I, Le Boulaire F, de Rose R, Samson F, Biaudet V, Lecharny A, Cruaud C, Weissenbach J, Caboche M, Lepiniec L (2001) Improved PCR-walking for large-scale isolation of plant T-DNA borders. Biotechniques 30:496–498PubMedGoogle Scholar
  3. Bec S, Chen L, Michaux Ferriere N, Legavre T, Fauquet C, Guiderdoni E (1998) Comparative histology of microprojectile-mediated gene transfer to embryogenic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability. Plant Sci 138:177–190CrossRefGoogle Scholar
  4. Devic M, Albert S, Delseny M, Roscoe TJ (1997) Efficient PCR walking on plant genomic DNA. Plant Physiol Biochem 35:331–339Google Scholar
  5. Droc G, Ruiz M, Larmande P, Pereira A, Piffanelli P, Morel JB, Dievart A, Courtois B, Guiderdoni E, Perin C (2006) OryGenesDB: a database for rice reverse genetics. Nucl Acids Res 34:D736–D740CrossRefPubMedGoogle Scholar
  6. Fukui K (1983) Sequential occurrence of mutations in a growing rice callus. Theor Appl Genet 65:225–230CrossRefGoogle Scholar
  7. Guiderdoni E, An G, Yu SM, Hsing YI, Wu C (2007) T-DNA insertion mutants as a resource for rice functional genomics. In: Upadhyaya NM (ed) Rice functional genomics- challenges, progress and prospects, Springer, NY, 187–227Google Scholar
  8. Hirochika H (2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4:118–122CrossRefPubMedGoogle Scholar
  9. Hirochika H, Guiderdoni E, An G, Hsing YI, Eun MY, Han CD, Upadhyaya N, Ramachandran S, Zhang QF, Pereira A, Sundaresan V, Leung H (2004) Rice mutant resources for gene discovery. Plant Mol Biol 54:325–334CrossRefPubMedGoogle Scholar
  10. Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M (1996) Retrotransposons of rice involved in mutations induced by tissue culture. PNAS 93:7783–7788CrossRefPubMedGoogle Scholar
  11. Hsing YI, Chern CG, Fan MJ, Lu PC, Chen KT, Lo SF, Sun PK, Ho SL, Lee KW, Wang YC, Huang WL, Ko SS, Chen S, Chen JL, Chung CI, Lin YC, Hour AL, Wang YW, Chang YC, Tsai MW, Lin YS, Chen YC, Yen HM, Li CP, Wey CK, Tseng CS, Lai MH, Huang SC, Chen LJ, Yu SM (2007) A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Mol Biol 63:351–364CrossRefPubMedGoogle Scholar
  12. Jeong DH, An S, Park S, Kang HG, Park GG, Kim SR, Sim J, Kim YO, Kim MK, Kim SR, Kim J, Shin M, Jung M, An G (2006) Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J 45:123–132CrossRefPubMedGoogle Scholar
  13. Jung KH, Han MJ, Lee YS, Kim YW, Hwang IW, Kim MJ, Kim YK, Nahm BH, An GH (2005) Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17:2705–2722CrossRefPubMedGoogle Scholar
  14. Matsumoto T, Wu JZ, Kanamori H, Katayose Y, Fujisawa M, Namiki N, Mizuno H, Yamamoto K, Antonio BA, Baba T, Sakata K, Nagamura Y, Aoki H, Arikawa K, Arita K, Bito T, Chiden Y, Fujitsuka N, Fukunaka R, Hamada M, Harada C, Hayashi A, Hijishita S, Honda M, Hosokawa S, Ichikawa Y, Idonuma A, Iijima M, Ikeda M, Ikeno M, Ito K, Ito S, Ito T, Ito Y, Ito Y, Iwabuchi A, Kamiya K, Karasawa W, Kurita K, Katagiri S, Kikuta A, Kobayashi H, Kobayashi N, Machita K, Maehara T, Masukawa M, Mizubayashi T, Mukai Y, Nagasaki H, Nagata Y, Naito S, Nakashima M, Nakama Y, Nakamichi Y, Nakamura M, Meguro A, Negishi M, Ohta I, Ohta T, Okamoto M, Ono N, Saji S, Sakaguchi M, Sakai K, Shibata M, Shimokawa T, Song JY, Takazaki Y, Terasawa K, Tsugane M, Tsuji K, Ueda S, Waki K, Yamagata H, Yamamoto M, Yamamoto S, Yamane H, Yoshiki S, Yoshihara R, Yukawa K, Zhong HS, Yano M, Sasaki T, Yuan QP, Shu OT, Liu J, Jones KM, Gansberger K, Moffat K, Hill J, Bera J, Fadrosh D, Jin SH, Johri S, Kim M, Overton L, Reardon M, Tsitrin T, Vuong H, Weaver B, Ciecko A, Tallon L, Jackson J, Pai G, Van Aken S, Utterback T, Reidmuller S, Feldblyum T, Hsiao J, Zismann V, Iobst S, de Vazeille AR, Buell CR, Ying K, Li Y, Lu TT, Huang YC, Zhao Q, Feng Q, Zhang L, Zhu JJ, Weng QJ, Mu J, Lu YQ, Fan DL, Liu YL, Guan JP, Zhang YJ, Yu SL, Liu XH, Zhang Y, Hong GF, Han B, Choisne N, Demange N, Orjeda G, Samain S, Cattolico L, Pelletier E, Couloux A, Segurens B, Wincker P, D'Hont A, Scarpelli C, Weissenbach J, Salanoubat M, Quetier F, Yu Y, Kim HR, Rambo T, Currie J, Collura K, Luo MZ, Yang TJ, Ammiraju JSS, Engler F, Soderlund C, Wing RA, Palmer LE, de la Bastide M, Spiegel L, Nascimento L, Zutavern T, O'Shaughnessy A, Dike S, Dedhia N, Preston R, Balija V, McCombie WR, Chow TY, Chen HH, Chung MC, Chen CS, Shaw JF, Wu HP, Hsiao KJ, Chao YT, Chu MK, Cheng CH, Hour AL, Lee PF, Lin SJ, Lin YC, Liou JY, Liu SM, Hsing YI, Raghuvanshi S, Mohanty A, Bharti AK, Gaur A, Gupta V, Kumar D, Ravi V, Vij S, Kapur A, Khurana P, Khurana P, Khurana JP, Tyagi AK, Gaikwad K, Singh A, Dalal V, Srivastava S, Dixit A, Pal AK, Ghazi IA, Yadav M, Pandit A, Bhargava A, Sureshbabu K, Batra K, Sharma TR, Mohapatra T, Singh NK, Messing J, Nelson AB, Fuks G, Kavchok S, Keizer G, Llaca ELV, Song RT, Tanyolac B, Young S, Il KH, Hahn JH, Sangsakoo G, Vanavichit A, de Mattos LAT, Zimmer PD, Malone G, Dellagostin O, de Oliveira AC, Bevan M, Bancroft I, Minx P, Cordum H, Wilson R, Cheng ZK, Jin WW, Jiang JM, Leong SA, Iwama H, Gojobori T, Itoh T, Niimura Y, Fujii Y, Habara T, Sakai H, Sato Y, Wilson G, Kumar K, McCouch S, Juretic N, Hoen D, Wright S, Bruskiewich R, Bureau T, Miyao A, Hirochika H, Nishikawa T, Kadowaki K, Sugiura M, Project IRGS, (2005) The map-based sequence of the rice genome. Nature 436:793–800CrossRefGoogle Scholar
  15. Miyao A, Iwasaki Y, Kitano H, Itoh J, Maekawa M, Murata K, Yatou O, Nagato Y, Hirochika H (2007) A large-scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes. Plant Mol Biol 63:625–635CrossRefPubMedGoogle Scholar
  16. Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 15:1771–1780CrossRefPubMedGoogle Scholar
  17. Moon S, Jung KH, Lee DE, Lee DY, Lee J, An K, Kang HG, An G (2006) The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size. Mol Cells 21:147–52PubMedGoogle Scholar
  18. Sallaud C, Gay C, Larmande P, Bes M, Piffanelli P, Piegu B, Droc G, Regad F, Bourgeois E, Meynard D, Perin C, Sabau X, Ghesquiere A, Glaszmann JC, Delseny M, Guiderdoni E (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39:450–464CrossRefPubMedGoogle Scholar
  19. Sallaud C, Meynard D, van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PB, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics. Theor Appl Genet 106:1396–408PubMedGoogle Scholar
  20. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucl Acids Res 23:1087–1088CrossRefPubMedGoogle Scholar
  21. Zhang J, Li C, Wu C, Xiong L, Chen G, Zhang Q, Wang S (2006) RMD: a rice mutant database for functional analysis of the rice genome. Nucleic Acids Res 34:D745–D748CrossRefPubMedGoogle Scholar
  22. Zhu QH, Eun MY, Han CD, Kumar CS, Pereira A, Ramachandran S, Sundaresan V, Eamens AL, Upadhyaya NM, Wu R (2007) Transposon insertional mutants: a resource for rice functional genomics. In: Upadhyaya NM (ed) Rice functional genomics- challenges, progress and prospects. Springer, NY, pp 229–280Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Pietro Piffanelli
    • 1
  • Gaétan Droc
    • 1
  • Delphine Mieulet
    • 1
  • Nadège Lanau
    • 1
  • Martine Bès
    • 1
  • Emmanuelle Bourgeois
    • 1
  • Claire Rouvière
    • 1
  • Fréderick Gavory
    • 2
  • Corinne Cruaud
    • 2
  • Alain Ghesquière
    • 3
  • Emmanuel Guiderdoni
    • 1
    Email author
  1. 1.Biological Systems Department TAA96/03CIRAD, UMR DAP 1098Montpellier cedex 5France
  2. 2.GénoscopeCentre National de SéquençageEvry cedexFrance
  3. 3.IRD, UMR GDP 5096Montpellier cedex 5France

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