Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus

Huang, Huibin; Cui, Tingting; Zhang, Lili; Yang, Qingyong; Yang, Yang; Xie, Kabin; Fan, Chuchuan; Zhou, Yongming

doi:10.1007/s00122-020-03607-y

Cited by 96 publications

(66 citation statements)

References 30 publications

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“…Classical EMS chemical mutagenesis was successfully used by Wells et al [ 19 ] and Bai et al [ 20 ] which is in common with our mutated materials [ 8 , 9 ]. However, more sophisticated methods, like clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated genome editing [ 21 ] or expression of RNA interference (RNAi) constructs, were also used for generation of gene-specific mutations [ 22 , 23 ] or to perform seed-specific silencing of Bna.FAD2 genes [ 24 ], respectively. To identify and localize the specific mutations in the Bna.FAD2 genes from our materials, both mutants (HOR3-M10453 and HOR4-M10464) and wild-type forms of rapeseed were studied at INRA, Le Rheu, France [ 25 – 27 ].…”

Section: Introductionmentioning

confidence: 99%

“…It was then confirmed that the mutations occurred in the Bna.FAD2 gene. To date, four copies of Bna.FAD2 gene were identified in the allotetraploid B. napus genome and the authors use different naming conventions to denote them [ 19 , 23 , 28 , 29 ]. The genes originating from Brassica rapa genome are localized on the A5 and A1 chromosomes and the genes originating from Brassica oleracea genome are localized on the C5 and C1 chromosomes.…”

Section: Introductionmentioning

confidence: 99%

“…All these genes were found to be transcriptionally active [ 29 ], but their expression profiles are different. While the mRNA products coming from the genes on the A5 and C5 chromosomes were present in all tissues, the mRNA products coming from the genes on the A1 and C1 chromosomes were specific for the developing seeds [ 23 , 29 ]. The observed level of transcript for the A5 copy of the Bna.FAD2 gene was the highest among all copies (especially during the seed development), and for the A1 and C1 copies this level was very low [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

“…While the mRNA products coming from the genes on the A5 and C5 chromosomes were present in all tissues, the mRNA products coming from the genes on the A1 and C1 chromosomes were specific for the developing seeds [ 23 , 29 ]. The observed level of transcript for the A5 copy of the Bna.FAD2 gene was the highest among all copies (especially during the seed development), and for the A1 and C1 copies this level was very low [ 23 ]. Finally, the gene on the chromosome A1 seems to be non-functional due to some deletions and insertions that result in the frameshift during translation and in effect the shortened protein is produced [ 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

“…The sequence alignment of studied clones with sequences from B. rapa (genome A) and B. oleracea (genome C) proved that both mutations are localized in the BnaA.FAD2 gene of B. napus (genome A) [ 25 ] (the gene nomenclature follows the rules proposed by Østergaard and King [ 12 ]). The previous reports on the non-functionality of the gene coming from the A1 chromosome suggest that the affected gene with the strong effect on the level of oleic acid in seeds must be the one localized on the A5 chromosome—denoted as BnFAD2-1 [ 29 ], BnaA.FAD2.a [ 19 , 28 ] or BnaFAD2.A5 [ 23 ]. The sequence analyses showed that the modified oleic acid content in the two studied mutants is due to the distinct changes in BnaA.FAD2 gene coding sequence.…”

Section: Introductionmentioning

confidence: 99%

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Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

et al. 2020

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Two mutants of winter rapeseed (Brassica napus L. var. oleifera) with an increased amount of oleic acid in seeds were created by chemical mutagenesis (HOR3-M10453 and HOR4-M10464). The overall performance of the mutated plants was much lower than that of wild-type cultivars. Multiple rounds of crossing with high-yielding double-low (“00”) cultivars and breeding lines having valuable agronomic traits, followed by selection of high oleic acid genotypes is then needed to obtain new “00” varieties of rapeseed having high oleic acid content in seeds. To perform such selection, the specific codominant cleaved amplified polymorphic sequences (CAPS) marker was used. This marker was designed to detect the presence of two relevant point mutations in the desaturase gene BnaA.FAD2, and it was previously described and patented. The specific polymerase chain reaction product (732 bp) was digested using FspBI restriction enzyme that recognizes the 5′-C↓TAG-3′ sequence which is common to both mutated alleles, thereby yielding band patterns specific for those alleles. The method proposed in the patent was redesigned, adjusted to specific laboratory conditions, and thoroughly tested. Different DNA extraction protocols were tested to optimize the procedure. Two variants of the CAPS method (with and without purification of amplified product) were considered to choose the best option. In addition, the ability of the studied marker to detect heterozygosity in the BnaA.FAD2 locus was also tested. Finally, we also presented some examples for the use of the new CAPS marker in the marker-assisted selection (MAS) during our breeding programs. The standard CTAB method of DNA extraction and the simplified, two-step (amplification/digestion) procedure for the CAPS marker are recommended. The marker was found to be useful for the detection of two mutated alleles of the studied BnaA.FAD2 desaturase gene and can potentially assure the breeders of the purity of their HOLL lines. However, it was also shown that it could not detect any other alleles or genes that were revealed to play a role in the regulation of oleic acid level.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

et al. 2020

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Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production

Khan

Zhu

et al. 2020

Journal Cellular Physiology

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Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T 1 and T 2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T 1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

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Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

2023

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Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome‐editing platforms employ sequence‐specific nucleases (SSNs) to generate DNA double‐stranded breaks at their target gene. Such DNA breaks are typically repaired via the error‐prone nonhomologous end‐joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end‐use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN‐mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

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Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus

Cited by 96 publications

References 30 publications

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Cleaved amplified polymorphic sequences (CAPS) marker for identification of two mutant alleles of the rapeseed BnaA.FAD2 gene

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

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