Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus.
Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, 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, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As 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.
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
Dorian Fabian
DF
Urbański
Anna
A
Małolepszy
Jens
J
Stougaard
Stig Uggerhøj
SU
Andersen
D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics; D018745:Genome, Plant / Q000235:genetics*; D060005:Genotyping Techniques / Q000379:methods*; D057166:High-Throughput Screening Assays; D000070116:Lotus / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D018626:Retroelements / Q000235:genetics*; D059386:Reverse Genetics; D012639:Seeds / Q000235:genetics; D017422:Sequence Analysis, DNA; D012984:Software; D020079:Terminal Repeat Sequences / Q000235:genetics
D017931:DNA Primers; D018626:Retroelements