Małolepszy Anna, Mun Terry, Sandal Niels, Gupta Vikas, Dubin Manu, Urbański Dorian, Shah Niraj, Bachmann Asger, Fukai Eigo, Hirakawa Hideki, Tabata Satoshi, Nadzieja Marcin, Markmann Katharina, Su Junyi, Umehara Yosuke, Soyano Takashi, Miyahara Akira, Sato Shusei, Hayashi Makoto, Stougaard Jens, Andersen Stig U
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK-8000, Aarhus C, Denmark.
Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030, Vienna, Austria.
Plant J. 2016 Oct;88(2):306-317. doi: 10.1111/tpj.13243. Epub 2016 Sep 27.
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.
长末端重复序列(LTR)逆转座子与逆转录病毒密切相关,它们的活动塑造了真核生物基因组。在此,我们展示了一个完整的日本百脉根插入突变体库,该库通过鉴定LTR元件百脉根逆转座子1(LORE1)(http://lotus.au.dk)从头激活后产生的640653个新插入事件而生成。插入偏好对于有效的基因靶向至关重要,我们利用这个庞大的数据集在此背景下分析LTR元件的特征。我们推断了产生逆转录病毒和LTR逆转座子插入位点典型共有回文序列的机制,鉴定出一个短的宽松插入位点基序,并证明其选择性整合到CHG低甲基化基因中。这些特征导致激活后有害突变率急剧增加,并使LORE1活性基因靶向在134682个日本百脉根株系群体中接近饱和。我们建议,使用具有生殖活性的内源性LTR逆转座子进行饱和诱变可作为一种通用且经济高效的策略,用于生成非转基因突变体库,供植物研究无限制使用。