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Abstract
Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.
Highlights
The discovery and utilization of lethal genes is of great significance to the progress of molecular breeding
As a powerful tool for genome editing, the CRISPR-Cas9 system was first successfully applied to plants by editing an albino gene OsPDS in rice, which exhibited an expected phenotype in T0 generation [1]
Using the zb7 gene as an editing target, a large number of lethal seedlings observed in T0 generation, which proved that the dmc1 promotercontrolled (DPC) CRISPR/Cas9 is a high-efficiency editing system in maize [2]
Summary
The discovery and utilization of lethal genes is of great significance to the progress of molecular breeding. As a powerful tool for genome editing, the CRISPR-Cas system was first successfully applied to plants by editing an albino gene OsPDS in rice, which exhibited an expected phenotype in T0 generation [1]. Using the zb gene as an editing target, a large number of lethal seedlings observed in T0 generation, which proved that the dmc promotercontrolled (DPC) CRISPR/Cas is a high-efficiency editing system in maize [2]. Few studies about gene cloning for lethality were reported for rapeseed due to the complex genome of Brassica napus (B. napus) and the limited reference genome before the release of B. napus, “ZS11”. The functional analysis of lethal mutants in rapeseed has potential value for improving gene editing efficiency and advancing technical innovation in molecular breeding
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