Why old duplicated genes are not thrown away in (paleo)polyploids? Example from the petC gene in Brassica napus.

Duplication of genes at different time period, through recurrent and frequent polyploidization events, have played a major role in plant evolution, adaptation and diversification. Interestingly, some of the ancestral duplicated genes (referred as paleologs), have been maintained for millions of years, and there is still a poor knowledge of the reasons of their retention, especially when testing the phenotypic effect of individual copies by using functional genetic approaches. To fill this gap, we performed functional genetic (CRISPR-Cas9), physiological, transcriptomic and evolutionary studies to finely investigate this open question, taking the example of the petC gene (involved in cytochrome b6/f and thus impacting photosynthesis) that is present in four paleologous copies in the oilseed crop Brassica napus. RNA-Seq and selective pressure analyses suggested that all paleologous copies conserved the same function and that they were all highly transcribed. Thereafter, the Knock Out (K.O.) of one, several or all petC copies highlighted that all paleologous copies have to be K.O. to suppress the gene function. In addition, we could determine that phenotypic effects in single and double mutants could only be deciphered in high light conditions. Interestingly, we did not detect any significant differences between single mutants K.O. for either the A03 or A09 copy (despite being differentially transcribed), or even between mutants for a single or two petC copies. Altogether, this work revealed that petC paleologs have retained their ancestral function and that the retention of these copies is explained by their compensatory role, especially in optimal environmental conditions.

I-SceI and customized meganucleases-mediated genome editing in tomato and oilseed rape.

Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.