Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

TY - JOUR

T1 - Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

AU - Urbanski, Dorian Fabian

AU - Malolepszy, Anna

AU - Stougaard, Jens

AU - Andersen, Stig Uggerhøj

PY - 2012/2

Y1 - 2012/2

N2 - Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis and insertion site identification. So far, only floral dip T-DNA transformation of Arabidopsishas produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols including software for automated data analysis, which take full advantage of high next-generation sequencing throughput. Here we address these challenges by developing the FSTpoolit protocol and software package and we demonstrateits efficacy by detecting 8,935 LORE1 insertions in 3,744 Lotus japonicus plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1mutagenesis combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing, and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

AB - Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis and insertion site identification. So far, only floral dip T-DNA transformation of Arabidopsishas produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols including software for automated data analysis, which take full advantage of high next-generation sequencing throughput. Here we address these challenges by developing the FSTpoolit protocol and software package and we demonstrateits efficacy by detecting 8,935 LORE1 insertions in 3,744 Lotus japonicus plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1mutagenesis combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing, and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

KW - LORE1

KW - FSTpoolit

KW - insertion mutagenesis

KW - reverse genetics

KW - FST

KW - retrotransposon

U2 - 10.1111/j.1365-313X.2011.04827.x

DO - 10.1111/j.1365-313X.2011.04827.x

M3 - Journal article

C2 - 22014280

SN - 0960-7412

VL - 69

SP - 731

EP - 741

JO - Plant Journal

JF - Plant Journal

IS - 4

ER -