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

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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 Arabidopsis
has 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 demonstrate
its 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 LORE1
mutagenesis 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.
OriginalsprogEngelsk
TidsskriftPlant Journal
Vol/bind69
Nummer4
Sider (fra-til)731-741
Antal sider11
ISSN0960-7412
DOI
StatusUdgivet - feb. 2012

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