J Integr Plant Biol. ›› 2023, Vol. 65 ›› Issue (10): 2242-2261.DOI: 10.1111/jipb.13555

• New Technology • Previous Articles     Next Articles

Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants

Pavel Merkulov1,2, Sofya Gvaramiya1, Maxim Dudnikov1,2, Roman Komakhin1, Murad Omarov1, Alina Kocheshkova1, Zakhar Konstantinov1, Alexander Soloviev1,3,4, Gennady Karlov1, Mikhail Divashuk1 and Ilya Kirov1,2*   

  1. 1. All‐Russia Research Institute of Agricultural Biotechnology, Moscow 127550, Russia;
    2. Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia;
    3. All‐Russia Center for Plant Quarantine, Ramenski 140150, Russia;
    4. N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences, Moscow 127276, Russia
    *Correspondence: Ilya Kirov (kirovez@gmail.com)
  • Received:2023-04-04 Accepted:2023-08-08 Online:2023-08-09 Published:2023-10-01

Abstract: Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ (EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40×sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.

Key words: Arabidopsis, DNA methylation, Nanopore sequencing, transposon insertions

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