Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

Ahmar, Sunny; Zhai, Yungu; Huang, Huibin; Yu, Kaidi; Khan, Muhammad Hafeez Ullah; Shahid, Muhammad; Samad, Rana Abdul; Khan, Shahid Ullah; Amoo, Olalekan; Fan, Chuchuan; Zhou, Yongming

doi:10.1016/j.cj.2021.03.023

Cited by 20 publications

(12 citation statements)

References 40 publications

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Section: Discussioncontrasting

confidence: 57%

“…This study shows the successful utilization of CRISPR/Cas9 for targeted mutations of the BnaCRTISO gene in rapeseed with an editing efficiency of 75.00%, which is higher than those reported in previous research (14.4–45.0%) ( Yang et al, 2017 ; Zhai et al, 2019 ; Ahmar et al, 2021 ). The editing efficiency at the S2 target site (73.22%) is higher than S1 (16.39%), indicating that the efficiency of the sgRNA promoter U3b is higher than that of U3d as our previous reports ( Yang et al, 2017 ; Zhai et al, 2019 ; Ahmar et al, 2021 ). In this study, the targeted mutations of the two functional copies of BnaCRTISO in rapeseed generated the creamy white petal phenotype ( Supplementary Table 2 and Figure 2 ), indicating that the BnaCRTISO gene is essential for flower color regulation and highly conservative in Brassica plants.…”

Section: Discussioncontrasting

confidence: 57%

“…In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be an amazing tool for improving crops via site-specific genome editing, and CRISPR/Cas9 is considered the most simple and efficient SSN. The CRISPR/Cas9 system has been effectively utilized in rapeseed to produce the targeted mutations for the improvement of numerous agronomic traits ( Braatz et al, 2017 ; Yang et al, 2017 , 2018 ; Hu et al, 2018 ; Li C. et al, 2018 ; Zhai et al, 2019 ; Ahmar et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

Amoo

et al. 2022

Front. Plant Sci.

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The diversity of petal and leaf color can improve the ornamental value of rapeseed and promote the development of agriculture and tourism. The two copies of carotenoid isomerase gene (BnaCRTISO) in Brassica napus (BnaA09.CRTISO and BnaC08.CRTISO) was edited using the CRISPR/Cas9 system in the present study. The mutation phenotype of creamy white petals and yellowish leaves could be recovered only in targeted mutants of both BnaCRTISO functional copies, indicating that the redundant roles of BnaA09.CRTISO and BnaC08.CRTISO are vital for the regulation of petal and leaf color. The carotenoid content in the petals and leaves of the BnaCRTISO double mutant was significantly reduced. The chalcone content, a vital substance that makes up the yellow color, also decreased significantly in petals. Whereas, the contents of some carotenes (lycopene, α-carotene, γ-carotene) were increased significantly in petals. Further, transcriptome analysis showed that the targeted mutation of BnaCRTISO resulted in the significant down-regulation of important genes BnaPSY and BnaC4H in the carotenoid and flavonoid synthesis pathways, respectively; however, the expression of other genes related to carotenes and xanthophylls synthesis, such as BnaPDS3, BnaZEP, BnaBCH1 and BCH2, was up-regulated. This indicates that the molecular mechanism regulating petal color variation in B. napus is more complicated than those reported in Arabidopsis and other Brassica species. These results provide insight into the molecular mechanisms underlying flower color variation in rapeseed and provides valuable resources for rapeseed breeding.

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Section: Discussioncontrasting

confidence: 57%

Section: Discussioncontrasting

confidence: 57%

Section: Introductionmentioning

confidence: 99%

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Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

Amoo

et al. 2022

Front. Plant Sci.

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“…In our previous studies, the highly efficient genome editing platform has been established to dissect the functional diversity of different homologs in oilseed rape ( Li et al, 2018 ; Zaman et al, 2019 ; Cheng et al, 2021 ). Many independent case studies suggested that the homologous gene copies, although possess similar genomic information, often exert different effects in a particular trait ( Okuzaki et al, 2018 ; Zhai et al, 2019 , 2020 ; Ahmar et al, 2021 ; Chen et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Functional Differentiation of BnVTE4 Gene Homologous Copies in α-Tocopherol Biosynthesis Revealed by CRISPR/Cas9 Editing

et al. 2022

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Tocopherols are essential nutrients for human health known as vitamin E. Vitamin E deficiency can have a profound effect on human health, including the central nervous system and cardiovascular and immune protection. Multiple enzymatic steps are involved in the conversion between different forms of tocopherols. Among them, γ-tocopherol methyltransferase encoded by gene VTE4 catalyzes the conversion of γ- to α-tocopherol or δ- to β-tocopherol isoforms. However, the gene copies and their functional contribution of VTE4 homologs in Brassica napus were not elucidated. To this end, different mutation combinations of four putative BnVTE4 homologous copies were generated by using CRISPR/Cas9 genome editing technology. Editing of those BnVTE4 homologs led to a significant change of the α-tocopherol content and the ratio between α- and γ-tocopherol compared with wide-type control. Analysis of the different combinations of BnVTE4-edited homologs revealed that the contribution of the BnVTE4 individual gene displayed obvious functional differentiation in α-tocopherol biosynthesis. Their contribution could be in order of VTE4.C02-2 (BnaC02G0331100ZS) > VTE4.A02-1 (BnaA02G0247300ZS) > VTE4.A02-2 (BnaA02G0154300ZS). Moreover, the VTE4.A02-1 and VTE4.A02-2 copies might have severe functional redundancies in α-tocopherol biosynthesis. Overall, this study systemically studied the different effects of BnVTE4 homologs, which provided a theoretical basis for breeding high α-tocopherol content oilseed rape.

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“…Thus, harnessing genetic diversity and modifying genomes precisely will be important for crop breeding programs. Until now, apart from the CBEs applied to rapeseed (Ahmar et al 2022 ; Wu et al 2020 ), precise genome editing (e.g., HDR-based gene targeting, ABEs, and prime editing) had not been achieved in Brassica , and most mutants were obtained via the NHEJ pathway. There is a need for the entry of scientists whose focus is on genome editing into the Brassica field to promote new technologies for precise genome editing.…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives

Zhang

et al. 2022

aBIOTECH

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Brassica species are a global source of nutrients and edible vegetable oil for humans. However, all commercially important Brassica crops underwent a whole-genome triplication event, hindering the development of functional genomics and breeding programs. Fortunately, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies, by allowing multiplex and precise genome engineering, have become valuable genome-editing tools and opened up new avenues for biotechnology. Here, we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing. We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements. Finally, we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops. Ongoing advancement in CRISPR/Cas technologies, in combination with other achievements, will play a significant role in the genetic improvement and molecular breeding of Brassica crops.

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Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

Cited by 20 publications

References 40 publications

Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

Functional Differentiation of BnVTE4 Gene Homologous Copies in α-Tocopherol Biosynthesis Revealed by CRISPR/Cas9 Editing

The application of CRISPR/Cas technologies to Brassica crops: current progress and future perspectives

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