Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice (Oryza sativa L.) Lines through CRISPR/Cas9 Based Mutagenesis of Wx and TGW6 and Proteomic Analysis of Anther

Yue Han,Babar Usman,Dengjie Luo,Fang Liu,Rongbai Li,Neng Zhao,Gul Nawaz

doi:10.3390/agronomy8120290

Yue Han, Babar Usman + Show 5 more

Open Access

https://doi.org/10.3390/agronomy8120290

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Abstract

Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T1 plants were re-edited but T2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC2F1. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD+-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.

Highlights

The rice (Oryza sativa L.) is an important widely adapted food crop and 20% of the world’s dietary energy supply which is feeding more than half of the world’s population and 3 billion people uptake rice daily [1,2]
The results of this study indicate that the CRISPR/Cas9 gene editing technology can successfully edit rice targeting DNA sequences with high efficiency and multiple mutations can be generated at the same target site, and base deletion or insertion occurs before the target site protospacer adjacent motif (PAM)
The CRISPR/Cas9 technology induces fastest changes to plant genome than other molecular approaches and mutations passed to the generations without any rewriting or emendations

Summary

Introduction

The rice (Oryza sativa L.) is an important widely adapted food crop and 20% of the world’s dietary energy supply which is feeding more than half of the world’s population and 3 billion people uptake rice daily [1,2]. China is the pioneer of hybrid rice production and with the development of latest breeding tools the yield of rice has been increased more than 20% and newly developed genotypes performing better than conventional verities and accounts more than half of the annual rice planting area in China [6]. Yield and quality are typical quantitative traits governed by multiple genomic loci, while yield is directly depends on grain weight (GW) which is mainly determined by the synthesis and accumulation of starch in the endosperm of the grain [9,10]. To solve this problem, we must resort to new technologies and new genetic improvement strategies. Starch is one of the important indicators for evaluating rice quality and 90% of rice endosperm is starch [11]

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