Abstract
The Lotus Retrotransposon 1 (LORE1) is used for genome-wide mutagenesis of the model legume Lotus japonicus. Characterization of the LORE1 insertion sites in individual mutant lines is critical for development and use of the resource. Here we present guidelines for use of the LORE1 reverse genetics resource and provide detailed protocols for insertion site identification and validation. For high-throughput identification of insertions in up to 9,216 pooled lines, the FSTpoolit protocol takes advantage of Splinkerette adapters, molecular barcoding, 2D pooling, Illumina sequencing, and automated data analysis using the freely available FSTpoolit software. Complementing the high-throughput approach, we describe a simplified sequence-specific amplification polymorphism (SSAP) protocol well suited for quick identification of insertion sites in a limited number of lines. Both the FSTpoolit and simplified SSAP protocols are generally applicable to insertion site identification in any insertional mutagenesis setup.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Urbański DF, Małolepszy A, Stougaard J, Andersen SU (2012) Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J 69:731–741
Fukai E, Soyano T, Umehara Y, Nakayama S, Hirakawa H, Tabata S et al (2012) Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1. Plant J 69:720–730
Yokota K, Fukai E, Madsen LH, Jurkiewicz A, Rueda P, Radutoiu S et al (2009) Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti. Plant Cell 21:267–284
Stracke S, Kistner C, Yoshida S, Mulder L, Sato S, Kaneko T et al (2002) A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature 417:959–962
Kanamori N, Madsen LH, Radutoiu S, Frantescu M, Quistgaard EM, Miwa H et al (2006) A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci U S A 103:359–364
Madsen LH, Fukai E, Radutoiu S, Yost CK, Sandal N, Schauser L et al (2005) LORE1, an active low-copy-number TY3-gypsy retrotransposon family in the model legume Lotus japonicus. Plant J 44:372–381
Fukai E, Umehara Y, Sato S, Endo M, Kouchi H, Hayashi M et al (2010) Derepression of the plant chromovirus LORE1 induces germline transposition in regenerated plants. PLoS Genet 6:e1000868
Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K et al (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 15:1771–1780
D’Erfurth I, Cosson V, Eschstruth A, Lucas H, Kondorosi A, Ratet P (2003) Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula. Plant J 34:95–106
Sarni F, Grand C, Boudet AM (1984) Purification and properties of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase from poplar stems (Populus X euramericana). Eur J Biochem 139:259–265
Lacombe E, Hawkins S, Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O et al (1997) Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J 11:429–441
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
Hansen J, Jørgensen JE, Stougaard J, Marcker KA (1989) Hairy roots—a short cut to transgenic root nodules. Plant Cell Rep 8:12–15
Stougaard J (1995) Agrobacterium rhizogenes as a vector for transforming higher plants. Application in Lotus corniculatus transformation. Methods Mol Biol 49:49–61
Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2:487–496
Thykjær T, Schauser L, Danielsen D, Finneman J, Stougaard J (1998) Transgenic plants: agrobacterium-mediated transformation of the diploid legume Lotus japonicus. In: Celis JE (ed) Cell biology: a laboratory handbook, vol 3, 2nd edn. Academic, New York, pp 518–525
Devon RS, Porteous DJ, Brookes AJ (1995) Splinkerettes–improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res 23:1644–1645
Paithankar KR, Prasad KSN (1991) Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res 19:1346
Fukai E, Dobrowolska AD, Madsen LH, Madsen EB, Umehara Y, Kouchi H et al (2008) Transposition of a 600 thousand-year-old LTR retrotransposon in the model legume Lotus japonicus. Plant Mol Biol 68:653–663
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Urbański, D.F., Małolepszy, A., Stougaard, J., Andersen, S.U. (2013). High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants. In: Rose, R. (eds) Legume Genomics. Methods in Molecular Biology, vol 1069. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-613-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-613-9_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-612-2
Online ISBN: 978-1-62703-613-9
eBook Packages: Springer Protocols