Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L

Abstract

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.

Keywords: CRISPR/Cas9; Rf-like (RFL); cytological study; genome editing; rapeseed CMS; sgRNA.

Figure 1

Conserved motif analysis and phylogenetic relationship of BnaRFL11 with other gene species. (a) Phylogenetic tree showing an association between BnaRFL11 and its sister genes in Arabidopsis. Protein sequences were obtained from the GenBank with the following gene ID: BnaA09g45590D, AT1G12300, and AT1G12620. (b) Presence of similar motifs between sequences. (c,d) The conserved motifs among BnaRFL11, known fertility restore genes Rfn and Rfp, and Arabidopsis thaliana sister genes.

Figure 2

Model of BnaRFL11 with target sequences and the binary plasmid Cas9 vector. (a) The BnaRFL11 gene structure is shown in black. The vertical line indicates the gene model, and the arrow shows the sgRNA direction. The target sequences of sgRNAs are represented by PAM sites highlighted in red. (b) The kanamycin resistance cassette is driven by the CaMV35S promoter of the cauliflower mosaic virus. Two sgRNAs are driven by the U6-26p and U6-29p promoters and the U6-26t terminator.

Figure 3

Different stages of the tissue culture process for Agrobacterium-mediated transformation in rapeseed. (a) Sowing of wild-type Westar seeds on the seedling medium (M0). (b) Growth of plants on seedling medium after 6–7 days. (c) Spreading of hypocotyls for 48 h in the dark on (M1) media. (d) After 48 h, transfer explants were transferred to (M2) media and placed in a growth room for 15–20 days. (e) After 15–20 days, explants were on (M3) media for callus induction until the plants were regenerated. (f) Regenerated plants were transferred into (M4) rooting media to obtain better roots. (g) After obtaining roots, transgenic plants were transferred into pots under controlled conditions for their survival. (h) Transgenic plants were transferred to the field under normal conditions after survival. Bar = 4 cm.

Figure 4

Mutation transmission from T₀ to T₁ generations. At both target sites in bnarfl11 mutant plants. Red font and red hyphens signify CRISPR-based alterations, whereas PAM is underlined and highlighted in red. The altered alleles of RFL11 on chromosome A were expressed as aa09. “−” and “+” signify losses.

Figure 5

Phenotypic analysis of CRISPR/Cas9 mutated bnarfl11 plants. (a,b) Shows wild type with normal flower development. (c,d) Shows bnarfl11 flower.

Figure 6

Pollen viability test staining with 1% (w/v) acetocarmine solution. (a) Shows WT pollens. (b) Arrows indicating bnarfl11 degenerated pollens.

Figure 7

Plant growth stages of bnarfl11 compared to W.T. (a) Male-sterile plant at the close flower stage. (b) Male-sterile plant at the open-flower stage. (c) Male-sterile plants after crossing with the wild type with significantly fewer seeds. (d–f) Wild-type growth stages compared with those of bnarfl11.

Figure 8

Cytological analysis of bnarfl11 (i–p) compared to the wild type (a–h). There is no significant difference at early stages (a,b,i,j), but at later stages (c,d,k,l), the loss of one or two locules per anther is evident. In (l), MMCs are intermittently formed with an intact tapetum in the locks compared to W.T. Ar, archesporial cell; E, epidermis; PSC, primary sporogenous cell; MMC, microspore mother cells; S.P., sporogenous cell; En, endothecium; SPC, secondary parietal cell; ML, middle layer; T, tapetum; V, vascular region; PPC, primary parietal cell Bar = 10 μm. Cytological analysis of bnarfl11 compared to the wild type. (e–h) Stages of the wild type from 5–8. (m–p) displays the stages of the sterile flower from 5–8. (m) The Meiocyte stage with a highly dense callose wall around them and vacuolation in the tapetum was more confirmed than W.T. (e,n) At the tetrad stage, clumps of dense tissue in CMS locules, while in W.T. (f) locules and MC are typical. (o) At the pre-dehiscent stage, the locule increases in size, microspores form a cell clump, and the tapetum starts degrading; however, in W.T. (g), they have four large locules and normal microspores. (p) Degenerated pollen grains in the CMS flowers. (h) Normal pollen grains. T, tapetum; MC, meiotic cell; E, epidermis; St, stomium; Msp, microspores; S.P., sporogenous cell; Tds, tetrads; V, vascular region; P.G., pollen grain; DGP, degenerated pollen grains Bar = 10 μm.

Figure 8

Cytological analysis of bnarfl11 (i–p) compared to the wild type (a–h). There is no significant difference at early stages (a,b,i,j), but at later stages (c,d,k,l), the loss of one or two locules per anther is evident. In (l), MMCs are intermittently formed with an intact tapetum in the locks compared to W.T. Ar, archesporial cell; E, epidermis; PSC, primary sporogenous cell; MMC, microspore mother cells; S.P., sporogenous cell; En, endothecium; SPC, secondary parietal cell; ML, middle layer; T, tapetum; V, vascular region; PPC, primary parietal cell Bar = 10 μm. Cytological analysis of bnarfl11 compared to the wild type. (e–h) Stages of the wild type from 5–8. (m–p) displays the stages of the sterile flower from 5–8. (m) The Meiocyte stage with a highly dense callose wall around them and vacuolation in the tapetum was more confirmed than W.T. (e,n) At the tetrad stage, clumps of dense tissue in CMS locules, while in W.T. (f) locules and MC are typical. (o) At the pre-dehiscent stage, the locule increases in size, microspores form a cell clump, and the tapetum starts degrading; however, in W.T. (g), they have four large locules and normal microspores. (p) Degenerated pollen grains in the CMS flowers. (h) Normal pollen grains. T, tapetum; MC, meiotic cell; E, epidermis; St, stomium; Msp, microspores; S.P., sporogenous cell; Tds, tetrads; V, vascular region; P.G., pollen grain; DGP, degenerated pollen grains Bar = 10 μm.

Figure 9

Dehiscence stage of bnarfl11 compared with W.T. (a,b) bnarfl11 dehiscence stage. (a) Heap of scarring in exine (Ex) if four locks succeed in development. (b) The loss of adaxial or abaxial locules is evident in the bnarfl11. (c,d) Wild-type flowers at dehiscence, with four locules and normal mature pollen grains. Bar = 10 μm.

Figure 10

RT-PCR and qRT-PCR of bnarfl11 plants. (a) RT-PCR analysis of nap-CMS cytoplasm in bnarfl11 plants. For RT-PCR, total DNA was extracted from the Cas9 transgenic line, and the W.T. leaves. (b–f) qRT-PCR analysis of CMS-causing genes in bnarfl11 plants. For qRT-PCR analysis, total RNA was extracted from leaves, stems, buds, and petals of the Cas9 transgenic line. Values represent the mean and standard deviation. Error bars indicate the standard deviation among triplicate experiments.

Figure 11

Putative model for male sterility in the current study on rapeseed. Red arrow indicating upregulation.