Impact of differential DNA methylation on transgene expression in cotton (Gossypium hirsutumL.) events generated by targeted sequence insertion

First author: Aurine Verkest; Affiliations: BASF Agricultural Solutions Belgium: Gent, Belgium

Corresponding author: Kathleen D'Halluin

Targeted Genome Optimization (TGO) using site‐specific nucleases to introduce a DNA double‐strand break (DSB) at a specific target locus has broadened the options available to breeders for generation and combination of multiple traits. The use of targeted DNA cleavage in combination with homologous recombination (HR)‐mediated repair, enabled the precise targeted insertion of additional trait genes (2mepsps, hppd, axmi115) at a pre‐existing transgenic locus in cotton. Here we describe the expression and epigenome analyses of cotton Targeted Sequence Insertion (TSI) events over generations. In a subset of events, we observed variability in the level of transgene (hppd, axmi115) expression between independent but genetically identical TSI events. Transgene expression could also be differential within single events and variable over generations. This expression variability and silencing occurred independently of the transgene sequence and could be attributed to DNA methylation that was further linked to different DNA methylation mechanisms. The trigger(s) of transgene DNA methylation remains elusive but we hypothesize that targeted DSB induction and repair could be a potential trigger for DNA methylation.

定向基因组优化利用位点特异性核酸酶在特异性靶位点上引入DNA双链断裂DSB，这拓宽了育种研究中多个性状的组合培育。利用结合同源重组介导的修复的定向DNA剪切能够在棉花中一个已经存在的转基因位点上精确定向插入其它的性状基因（2mepsps、hppd和axmi115）。本文揭示了棉花定向序列插入TSI事件在几个世代后的表达和表观基因组。在一组事件中，作者在相互独立但遗传上一致的TSI事件间观察到了转基因（hppd和axmi115）表达的变化。转基因表达在单个事件中也会不同，世代间也会有变异。这种表达变异和沉默的发生是独立于转基因序列的，可能是由于DNA甲基化的作用，进一步的可能是因为不同的DNA甲基化机制所导致的。转基因DNA甲基化的诱发机制并不清楚，但是作者假设定向DSB诱导和修复可能诱导了DNA甲基化。

doi: https://doi.org/10.1111/pbi.13049

Journal: Plant Biotechnology Journal

First Published: 13 December, 2018