Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in <i>Brassica napus</i> L.
Zunaira Farooq,
Muhammad Nouman Riaz,
Muhammad Shoaib Farooq,
Yifan Li,
Huadong Wang,
Mayra Ahmad,
Jinxing Tu,
Chaozhi Ma,
Cheng Dai,
Jing Wen,
Jinxiong Shen,
Tingdong Fu,
Shouping Yang,
Benqi Wang,
Bin Yi
Affiliations
Zunaira Farooq
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Muhammad Nouman Riaz
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Muhammad Shoaib Farooq
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Yifan Li
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Huadong Wang
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Mayra Ahmad
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Jinxing Tu
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Chaozhi Ma
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Cheng Dai
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Jing Wen
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Jinxiong Shen
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Tingdong Fu
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Shouping Yang
Soybean Research Institute, National Center for Soybean Improvement, Key Laboratory of Biology and Genetic Improvement of Soybean (General Ministry of Agriculture), Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, China
Benqi Wang
Wuhan Vegetable Research Institute, Wuhan Academy of Agricultural Science and Technology, Wuhan 430065, China
Bin Yi
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement, Huazhong Agricultural University, Wuhan 430070, China
Brassica napus L. (canola, oil seed rape) is one of the world’s most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants’ vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study’s findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.
