Sensitive detection of pre-integration intermediates of long terminal repeat retrotransposons in crop plants

Retrotransposons have played an important role in the evolution of host genomes^1,2. Their impact is mainly deduced from the composition of DNA sequences that have been fixed over evolutionary time². Such studies provide important 'snapshots' reflecting the historical activities of transposons but do not predict current transposition potential. We previously reported sequence-independent retrotransposon trapping (SIRT) as a method that, by identification of extrachromosomal linear DNA (eclDNA), revealed the presence of active long terminal repeat (LTR) retrotransposons in Arabidopsis³. However, SIRT cannot be applied to large and transposon-rich genomes, as found in crop plants. We have developed an alternative approach named ALE-seq (amplification of LTR of eclDNAs followed by sequencing) for such situations. ALE-seq reveals sequences of 5' LTRs of eclDNAs after two-step amplification: in vitro transcription and subsequent reverse transcription. Using ALE-seq in rice, we detected eclDNAs for a novel Copia family LTR retrotransposon, Go-on, which is activated by heat stress. Sequencing of rice accessions revealed that Go-on has preferentially accumulated in Oryza sativa ssp. indica rice grown at higher temperatures. Furthermore, ALE-seq applied to tomato fruits identified a developmentally regulated Gypsy family of retrotransposons. A bioinformatic pipeline adapted for ALE-seq data analyses is used for the direct and reference-free annotation of new, active retroelements. This pipeline allows assessment of LTR retrotransposon activities in organisms for which genomic sequences and/or reference genomes are either unavailable or of low quality.