Advertisement

A User’s Guide to the Arabidopsis T-DNA Insertion Mutant Collections

  • Ronan C. O’Malley
  • Cesar C. Barragan
  • Joseph R. EckerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1284)

Abstract

The T-DNA sequence-indexed mutant collections contain insertional mutants for most Arabidopsis thaliana genes and have played an important role in plant biology research for almost two decades. By providing a large source of mutant alleles for in vivo characterization of gene function, this resource has been leveraged thousands of times to study a wide range of problems in plant biology. Our primary goal in this chapter is to provide a general guide to strategies for the effective use of the data and materials in these collections. To do this, we provide a general introduction to the T-DNA insertional sequence-indexed mutant collections with a focus on how best to use the available data sources for good line selection. As isolation of a homozygous line is a common next step once a potential disruption line has been identified, the second half of the chapter provides a step-by-step guide for the design and implementation of a T-DNA genotyping pipeline. Finally, we describe interpretation of genotyping results and include a troubleshooting section for common types of segregation distortions that we have observed. In this chapter we introduce both basic concepts and specific applications to both new and more experienced users of the collections for the design and implementation of small- to large-scale genotyping pipelines.

Key words

T-DNA Insertional mutagenesis T-DNA express SiGNAL SALK homozygous Plant genotyping High-throughput genotyping 

Notes

Acknowledgments

We would like to thank the entire National Science Foundation for funding of projects related to development of the Arabidopsis T-DNA insertion line resources (NSF MCB-1122250).

References

  1. 1.
    Rhee SY, Mutwil M (2014) Towards revealing the functions of all genes in plants. Trends Plant Sci 19:212–221CrossRefPubMedGoogle Scholar
  2. 2.
    Koboldt DC, Steinberg KM, Larson DE, Wilson RK (2013) The next-generation sequencing revolution and its impact on genomics. Cell 155:27–38CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Carvunis A-R, Ideker T (2014) Siri of the cell: what biology could learn from the iPhone. Cell 157:534–538CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846CrossRefPubMedGoogle Scholar
  6. 6.
    Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646CrossRefPubMedGoogle Scholar
  7. 7.
    Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Elling U, Taubenschmid J, Wirnsberger G, O’Malley R, Demers S-P, Vanhaelen Q, Shukalyuk AI, Schmauss G, Schramek D, Schnuetgen F et al (2011) Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9:563–574CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Kettleborough RNW, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, Sealy I, White RJ, Herd C, Nijman IJ et al (2013) A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 496:494–497CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Dietzl G, Chen D, Schnorrer F, Su K-C, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156CrossRefPubMedGoogle Scholar
  11. 11.
    O’Malley RC, Ecker JR (2010) Linking genotype to phenotype using the Arabidopsis unimutant collection. Plant J 61:928–940CrossRefPubMedGoogle Scholar
  12. 12.
    Tzfira T, Li J, Lacroix B, Citovsky V (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20:375–383CrossRefPubMedGoogle Scholar
  13. 13.
    Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  14. 14.
    Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C et al (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14:2985–2994CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657CrossRefPubMedGoogle Scholar
  16. 16.
    Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259CrossRefPubMedGoogle Scholar
  17. 17.
    Woody ST, Austin-Phillips S, Amasino RM (2007) The WiscDsLox T-DNA collection: an Arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline. J Plant Res 120:157–165CrossRefPubMedGoogle Scholar
  18. 18.
    Samson F, Brunaud V, Duchêne S, De Oliveira Y, Caboche M, Lecharny A, Aubourg S (2004) FLAGdb++: a database for the functional analysis of the Arabidopsis genome. Nucleic Acids Res 32Google Scholar
  19. 19.
    Sundaresan V, Springer P, Volpe T, Haward S, Jones JD, Dean C, Ma H, Martienssen R (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev 9:1797–1810CrossRefPubMedGoogle Scholar
  20. 20.
    Ito T, Motohashi R, Kuromori T, Mizukado S, Sakurai T, Kanahara H, Seki M, Shinozaki K (2002) A new resource of locally transposed dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome. Plant Physiol 129:1695–1699CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  22. 22.
    Wang YH (2009) How effective is T-DNA insertional mutagenesis in Arabidopsis? J Biochem Tech 1:11–20Google Scholar
  23. 23.
    O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat Protoc 2:2910–2917CrossRefPubMedGoogle Scholar
  24. 24.
    Clark KA, Krysan PJ (2010) Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines. Plant J 64:990–1001CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Nacry P, Camilleri C, Courtial B, Caboche M, Bouchez D (1998) Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149:641–650PubMedCentralPubMedGoogle Scholar
  26. 26.
    Lloyd J, Meinke D (2012) A comprehensive dataset of genes with a loss-of-function mutant phenotype in Arabidopsis. Plant Physiol 158:1115–1129CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ronan C. O’Malley
    • 1
    • 2
  • Cesar C. Barragan
    • 1
  • Joseph R. Ecker
    • 1
    • 2
    • 3
    Email author
  1. 1.Genomic Analysis LaboratorySalk Institute for Biological StudiesLa JollaUSA
  2. 2.Plant Biology LaboratorySalk Institute for Biological StudiesLa JollaUSA
  3. 3.Howard Hughes Medical InstituteNew YorkUSA

Personalised recommendations