High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants

Dorian Fabian Urbanski; Anna Malolepszy; Jens Stougaard; Stig Uggerhøj Andersen

doi:10.1007/978-1-62703-613-9_10

High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants

Dorian Fabian Urbanski, Anna Malolepszy, Jens Stougaard, Stig Uggerhøj Andersen

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

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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.

Original language	English
Journal	Methods in Molecular Biology
Volume	1069
Pages (from-to)	119-146
Number of pages	28
ISSN	1064-3745
DOIs	https://doi.org/10.1007/978-1-62703-613-9_10
Publication status	Published - 2013

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title = "High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants",

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.",

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doi = "10.1007/978-1-62703-613-9_10",

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T1 - High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants

AU - Urbanski, Dorian Fabian

AU - Malolepszy, Anna

AU - Stougaard, Jens

AU - Andersen, Stig Uggerhøj

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

U2 - 10.1007/978-1-62703-613-9_10

DO - 10.1007/978-1-62703-613-9_10

M3 - Journal article

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SN - 1064-3745

VL - 1069

SP - 119

EP - 146

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

ER -

High-Throughput and Targeted Genotyping of Lotus japonicus LORE1 Insertion Mutants

Abstract

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