The Sainsbury Laboratory, University of Cambridge, Cambridge, UK.
National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai, China.
Nat Plants. 2019 Jan;5(1):26-33. doi: 10.1038/s41477-018-0320-9. Epub 2018 Dec 10.
Retrotransposons have played an important role in the evolution of host genomes. Their impact is mainly deduced from the composition of DNA sequences that have been fixed over evolutionary time. 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 Arabidopsis. 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.
逆转录转座子在宿主基因组的进化中发挥了重要作用。它们的影响主要是根据在进化过程中固定的 DNA 序列组成来推断的。这些研究提供了重要的“快照”，反映了转座子的历史活动，但不能预测当前的转座潜力。我们之前报道了序列无关的逆转录转座子捕获（SIRT）作为一种方法，通过鉴定染色体外线性 DNA（eclDNA），揭示了拟南芥中活跃的长末端重复（LTR）逆转录转座子的存在。然而，SIRT 不能应用于作物等富含转座子的大型基因组。我们开发了一种替代方法，称为 ALE-seq（eclDNA 的 LTR 扩增和测序），用于这种情况。ALE-seq 通过两步扩增（体外转录和随后的反转录）揭示 eclDNA 的 5'LTR 序列：在体外转录和随后的反转录。在水稻中使用 ALE-seq，我们检测到了一个新的 Copia 家族 LTR 逆转录转座子 Go-on 的 eclDNA，它被热应激激活。对水稻品系的测序表明，Go-on 在生长在较高温度下的籼稻亚种中优先积累。此外，ALE-seq 在番茄果实中鉴定出一种发育调节的 Gypsy 家族逆转录转座子。一个适应 ALE-seq 数据分析的生物信息学管道用于新的、活跃的逆转录元件的直接和无参考注释。该管道允许评估基因组序列和/或参考基因组不可用或质量较低的生物体中 LTR 逆转录转座子的活性。
