The LORE1 insertion mutant resource

Summary Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG‐hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost‐efficient strategy for generation of non‐transgenic mutant collections for unrestricted use in plant research. Significance Statement Genome‐wide insertional inactivation of genes has enabled functional genomics in increasing numbers of plant species. By activating an endogenous LTR retrotransoposon in the legume Lotus japonicus and annotating a very large number of insertion sites, nearly all active genes were targeted by activation of just a single LTR element. This was made possible due to the relaxed insertion site motif and preference for CHG‐hypomethylated genes of the LTR element.