The TaQ alleles as one of the AP2-like transcription factors in common wheat (Triticum aestivum) play an important role in the evolution of spike characteristics from wild and domesticated emmer to modern wheat cultivars. Its loss-of-function mutant not only changed threshability and spike architecture but also affected plant height, flowering time, and floret structure. However, the comprehensive functions of TaAQ and TaDq genes in wheat have not been fully elucidated yet. Here, CRISPR/SpCas9 was used to edit wheat TaAQ and TaDq. We obtained homozygous plants in the T1 generation with loss of function of only TaAQ or TaDq and simultaneous loss of function of TaAQ and TaDq to analyze the effect of these genes on wheat spikes and floret shapes. The results demonstrated that the TaAQ-edited plants and the TaAQ and TaDq simultaneously-edited plants were nearly similar in spike architecture, whereas the TaDq-edited plants were different from the wild-type ones only in plant height. Moreover, the TaAQ-edited plants or the TaAQ and TaDq simultaneously-edited plants were more brittle than the wild-type and the TaDq-edited plants. Based on the expression profiling, we postulated that the VRN1, FUL2, SEP2, SEP5, and SEP6 genes might affect the number of spikelets and florets per spike in wheat by regulating the expression of TaQ. Combining the results of this report and previous reports, we conceived a regulatory network of wheat traits, including plant height, spike shape, and floral organs, which were influenced by AP2-like family genes. The results achieved in this study will help us to understand the regulating mechanisms of TaAQ and TaDq alleles on wheat floral organs and inflorescence development.
Grain production of cereal crops is closely associated with various botanic and agronomic traits, and improvement of these traits can contribute to yield increase. It has been clarified that plant height, spikelet density, and the inflorescence structure determine not only plant architecture but also grain yield (Wang and Li, 2008). Recent studies demonstrated that the modification of crop inflorescence development could increase grain number and size and influence threshability (Debernardi et al., 2017). Crop inflorescence development initiates once the shoot apical meristem shifts from the vegetative to the reproductive stage (McSteen et al., 2000). In some grass species, the inflorescence meristem produces primary and secondary branches for more spikelets (Poethig, 1990). In barley (Hordeum vulgare L.), rice (Oryza sativa L.), sorghum [Sorghum bicolor (L.) Moench], and maize (Zea mays L.), the number of florets was determined by the meristem, whereas in wheat (Triticum aestivum L.), the number of lateral florets is indeterminate, and only the basal florets can develop into grains (Sakuma et al., 2019).
In Arabidopsis, APETALA2 (AP2) is defined as a floral organ identity gene, which encodes a transcriptional factor. Five AP2-like genes TARGET OF EAT1 (TOE1), TOE2, TOE3, SCHLAFMUTZE (SMZ), and SCHNARCHZAPFEN (SNZ) primarily were demonstrated to act as flowering repressors (Jung et al., 2007). A loss-of-function mutant of INDETERMINATE SPIKELET1 (IDS1) and SISTER OF INDETERMINATE SPIKELET 1 (SID1) in maize, belonging to the members of AP2-like family, resulted in the spike architecture altered (Tang et al., 2007; Chuck et al., 2008). A combined loss-of-function mutant of OsIDS1 and SUPERNUMERARY BRACT (SNB) in rice resulted in multiple bract-like structures in spikelets before production of one or more florets (Hong et al., 2010; Lee and An, 2012). In wheat, the loss-of-function mutant of the AP2-like transcription factor, the Q gene, changed flowering time and spike architecture (Brown and Bregitzer, 2011; Varkonyi-Gasic et al., 2012).
Previous studies revealed that the conserved microRNA172 (miR172) targeted the AP2-like family and influenced the inflorescence and spikelet development in Arabidopsis, maize, and barley (Zhu and Helliwell, 2011). The expression of AP2-like transcription factor genes in Arabidopsis such as TOE1, TOE2, TOE3, SMZ, and SNZ was suppressed by miR172, and the expression of tasselseed4 (ts4) and the AP2-like gene IDS1 in maize was regulated by miR172. However, ts4 or IDS1 mutant containing a transposon insertion or a mutation at the miR172-binding site in maize showed abnormal differentiation of male or female organs (Chuck et al., 2007). A transposon insertion in miR172 led to development of abnormal spikelets and more florets in barley (Brown and Bregitzer, 2011). In wheat, overexpression of a miR172 precursor changed spike shape and caused sterility of florets (Debernardi et al., 2017).
Up to now, a few AP2-like genes of AP2L1, AP2L2, AP2L5, and AP2L7 were identified in wheat (Debernardi et al., 2020), among which AP2L5 was nominated as TaQ in the AP2 family. During the development of wheat flowers and spikes, AP2 was likely to regulate the development of spikes and florets by inhibiting the expression of related decisive genes (Aukerman and Sakai, 2003; Jung et al., 2007). Wheat TaQ alleles existing in most varieties have important roles in the domestication of spike architecture (Zhang et al., 2011). The loss-of-function mutant of TaQ gene not only changed threshability and spike phenotype but also affected plant height, flowering time, rachis fragility, glume toughness, and floral transition (Faris et al., 2005; Zhang et al., 2011; Debernardi et al., 2017). TaQ is an orthologue gene of IDS1 (Debernardi et al., 2020). Like other AP2-like genes, TaQ and its homoeologues have a miR172 target site in their coding regions, which influences mRNA stability (Simons et al., 2006; Debernardi et al., 2017). In tetraploid wheat (Triticum turgidum), mutation in the miR172 target site of TaQ alleles resulted in less targeting effectiveness, which further led to a conversion from glumes to florets in the spikelets in the distal positions of the spike. Moreover, the mutation affected spike development and contributed to free-threshing characteristics (Debernardi et al., 2017). The overexpression of TaQ reduced spike length, increased the floret number, and promoted conversion of glumes to lemma-like organs or even fertile florets (Song et al., 2019). In contrast, the overexpression of miR172 in transgenic wheat plants showed similar phenotypes to TaQ mutants and resulted in the formation of additional sterile florets with intermediate characteristics between glumes and lemmas (Debernardi et al., 2017).
Wheat as a polyploid species has three homologous TaQ genes. The homologous allele on the long arm of chromosome 5AL was well characterized (Kato et al., 2003; Simons et al., 2006). TaAQ is thought to be a major regulatory gene for floral development and has a pleiotropic function in wheat domestication (Muramatsu, 1986). The second homeologue TaBq on 5BL is a pseudogene. The third homeologue TaDq on 5DL expresses at quite a low level. The Q allele is incompletely dominant to q, and TaDq and TaBq together regulate the expression of TaAQ. When TaAQ is absent, TaDq also has an important role in controlling wheat spike shape (Zhang et al., 2011). Taq and TaQ loss-of-function mutant produced a speltoid-like spike phenotype and non–free-threshing grains, which were similar to the spikes of emmer wheat (Faris et al., 2005). Notably, Taq has a dosage effect, and a high dosage of Taq resulted in a square-headed spike (Muramatsu, 1963). Until now, the comprehensive functions of TaAQ and TaDq are yet to be determined, and little is known on the molecular mechanisms of TaQ and Taq in regulating wheat spike architecture.
Since the establishment of efficient wheat genetic transformation systems (Ishida et al., 2015; Zhang et al., 2015), the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in wheat to produce mutation of several genes (Jiao and Gao, 2016; Liang et al., 2017, 2018; Wang et al., 2018). For example, TaMLO-, TaEDR1-, and TaGW2-edited wheat plants showed increased levels of powdery mildew resistance and larger grain size (Wang et al., 2014; Zhang et al., 2017, 2018). In another example, low-gluten wheat lines were produced using the CRISPR-editing system, and the content of α-gliadins in seed kernels was significantly reduced (Sanchez-Leon et al., 2018). In addition, some groups have obtained wheat mutants using the Agrobacterium-mediated CRISPR/Cas9 system. Examples include successful prolonged seed dormancy in mutants by editing the threeTaQsd1 homeologues (Abe et al., 2019), haploid seed-producing progeny from the TaMTL/TaPLA gene-edited wheat (Liu et al., 2020a, 2020b), and increased grain number and weight per spike in TaCKX2-1 gene mutants (Zhang et al., 2019b).
In the present study, we used our aforementioned optimized Agrobacterium-mediated CRISPR/SpCas9 system to edit wheat TaAQ and TaDq to obtain alone and double knockout mutants (Liu et al., 2020b). We also analyzed the effects of TaAQ and TaDq on wheat spike shape and plant height. Finally, a network on various wheat botanic and agronomic traits including plant height, spike shape, and floral organs regulated by the genes in the AP2-like family was conceived. Our present study will be helpful to understand the molecular mechanisms of wheat spike architecture and inflorescence development modulated by the TaAQ and TaDq alleles.
2.
Results
2.1
Mutation of TaQ genes induced by the CRISPR/SpCas9 system in the T0-edited plants
A total of 112 and 75 T0 transgenic wheat plants derived from Fielder and Jimai 22 were obtained, respectively. The polymerase chain reaction-restriction enzyme (PCR-RE) assay confirmed that the TaQ-edited events occurred in 51 Fielder and 33 Jimai 22 plants, and the editing efficiencies were 45.6% and 44.0%, respectively (Fig. S2C and S2D; Table 1 and Table S3). Regarding the TaAQ target site, 32 Fielder- and 25 Jimai 22-edited plants were identified in the transformation experiments, with the editing efficiencies of 28.6% and 33.3%, respectively. Among the edited Fielder plants, nine were biallelic mutants and 23 were heterozygous mutants. Among the edited Jimai 22 plants, nine were biallelic mutants and 16 were heterozygous mutants. When confirming the TaDq target site, we obtained 29 Fielder- and 18 Jimai 22-edited plants, and the editing efficiencies were 25.9% and 24.0%, respectively. Among them, 10 and 8 plants were biallelic, and 19 and 10 plants were heterozygous from the two cultivars, respectively. Ten plants were simultaneously edited at the two targets of TaAQ and TaDq in Fielder individuals, and the editing efficiency was 8.9%; moreover, 4 plants were identified to be biallelic at the targets of TaAQ and TaDq. In Jimai 22, 10 plants were simultaneously edited at the two targets of TaAQ and TaDq, with an editing efficiency of 13.3%, and 3 plants were identified to be biallelic mutants at the targets of TaAQ and TaDq (Fig. S2C and D; Table 1 and S3). Sanger sequencing showed that 1- to 5-bp deletions at the 3 bp upstream of the PAM sites within the sgRNA were the primary knockout type for the TaAQ gene in the T0-edited plants, and a large DNA fragment of a 52-bp deletion was also identified for this gene. For the TaDq allele, 1-, 2-, 4-, and 5-bp deletions at the 3 bp upstream of the PAM sites within the sgRNA were identified in the T0 mutants (Fig. S3). These results demonstrate that the CRISPR/SpCas9 system works well in editing wheat TaQ genes.
Table
1.
Summary of the editing efficiency of TaQ in wheat varieties Fielder and Jimai 22 using the CRISPR/SpCas9 system.
2.2
Knockout of TaQ genes affected wheat spike morphogenesis in T0-edited plants
To primarily understand the biological function of TaQ genes in wheat, we investigated the gene effect on spike morphogenesis by comparing the spikes of TaAQ and TaDq simultaneously-edited T0 mutants (henceforth, described as TaQ-edited plants) with the spikes of the wild-type plants of Fielder and Jimai 22. The spike architecture of the edited plants was significantly altered (Fig. 1). The Fielder-edited plants exhibited two phenotypes, in which the seed setting rates were significantly lower than those in the wild-type plants (Fig. 1A–E). However, more florets per spikelet were observed in one Fielder mutant than in the wild-type plants, but most of them were sterile, and no seed was obtained from this phenotype (Fig. 1A). Another mutant produced longer spikes than the wild-type plants and showed a reduced glume angle and awn length and two more lemma-like organs in spikelets (Fig. 1D and E). The Jimai 22 mutants displayed a longer speltoid-like spike and internode phenotypes than the wild-type plants (Fig. 1F). In addition, the edited plants from Jimai 22 also had a reduced glume angle and awn length and two more lemma-like organs in spikelets compared with the wild-type plants (Fig. 1I and J). Particularly, the mutants from Fielder and Jimai 22 produced difficult-to-thresh grains.
Fig.
1.
Spike morphology of the TaQ-edited wheat plants in T0 generation at two weeks after flowering. A [1],B, and C: The wild-type Fielder. A [2],A [3],D and E: The TaQ-edited plants derived from Fielder. F [1],G, and H: The wild-type Jimai 22. F [2],I, and J: The TaQ-edited plants derived from Jimai 22.
The transverse section staining of the rachis from the Jimai 22 TaQ-edited plants with Safranin O/Fast Green showed that they contained more lignin and less cellulose in their cell walls than the wild-type Jimai 22 plants (Fig. 2A and B). Thereby, the spikes of the TaQ-edited plants were more brittle than the wild-type Jimai 22 plants. Morphology and anatomy of the transverse sections of the second glume in the central spikelets between the wild-type Jimai 22 plants and its edited plants were also different. The edited plants had stronger keels than the wild-type ones, and the transverse sections of the glume's lateral regions showed increased sclerenchyma cells than the wild-type Jimai 22 plants (Fig. 2C–F). All the aforementioned results indicate that TaQ genes play an important role in regulating wheat spike architecture.
Fig.
2.
Transverse sections of the rachis and the second glume from central spikelets of the T0 edited wheat plants at two weeks after flowering. A and B: The transverse sections of rachis were stained with Safranin O/Fast Green. C–F: The transverse sections of glume were stained with toluidine blue. A, C, and E: The wild-type Jimai 22. B, D, and F: The TaQ-edited Jimai 22 plants.
2.3
Knockout of TaQ genes resulted in pleiotropic effects on botanic traits and modulated the expression of floral organ development-related genes in T1 plants
TaAQ-edited plants (Xe53-91-7, Xe53-17-2, and Xe53-25-9), TaDq-edited plants (Xe53-38-4 and Xe53-55-11), andTaAQ and TaDq simultaneously-edited plants (Xe53-8-2 and Xe53-13-4) derived from Fielder were selected to comprehensively compare the effects of the TaAQ and TaDq alleles on phenotypes of wheat plants. Plants Xe53-91-7, Xe53-17-2, and Xe53-25-9 had 3-bp, 5-bp, and 2-bp deletions in the PAM sites of the TaAQ sgRNA sequence, respectively. Plants Xe53-38-4 and Xe53-55-11 had 1-bp and 4-bp deletions in the PAM sites of the TaDq sgRNA sequence, respectively. Plant Xe53-8-2 had a 4-bp deletion in the TaAQ sgRNA sequence and a 1-bp deletion in the TaDq sgRNA sequence, and plant Xe53-13-4 had a 2-bp deletion in the TaAQ sgRNA sequence and a 4-bp deletion in the TaDq sgRNA sequence. All these plants in T1 generation were homozygous mutants (Fig. S4). In addition, the edited plants Xe53-91-7, Xe53-17-2, Xe53-38-4, Xe53-55-11, and Xe53-13-4 contained the selection marker of the bar gene, while the edited plants Xe53-25-9 and Xe53-8-2 were transgene free (Fig. S5).
The expression levels of TaQ were notably reduced in mutants Xe53-17-2, Xe53-25-9, Xe53-8-2, and Xe53-13-4 (Fig. S6A). The spikes in the TaAQ-edited plants Xe53-17-2 and Xe53-25-9 and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4 were speltoid-like spikes. Their spikelet and seed number per spike were reduced, and most of their spikelets carried less than one seed. In addition, the edited plants Xe53-17-2, Xe53-25-9, Xe53-8-2, and Xe53-13-4 showed longer spikes, shorter awns, and smaller glume angles than the wild-type Fielder plants (Fig.3 and S6). However, the spike shape of the edited plants Xe53-91-7, Xe53-38-4, and Xe53-55-11 was similar to that of the wild-type Fielder plants (Fig. 3). Plant height was significantly shorter in the edited plants Xe53-38-4 and Xe53-55-11 than in the wild-type plants (Fig. S6B). The results demonstrate that the TaAQ-edited plants and the TaAQ and TaDq simultaneously-edited plants are almost the same in spikelet and seed number per spike, glume angle, and spike and awn length, whereas the TaDq-edited plants are different from the wild-type plants only in plant height, and the plant height and spike shape of mutant Xe53-91-7 are still similar to the wild-type plant although the SpCas9 gene exists and a 3-bp deletion in the sgRNA sequence of TaAQ occurs in this mutant (Fig. 3 and S6).
Fig.
3.
Spike morphology of the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited wheat plants in T1 generation at two weeks after flowering. Samples 1 to 8 were the wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively.
The transverse section of the stained plant rachis with Safranin O/Fast Green revealed that the edited plants Xe53-91-7, Xe53-38-4, and Xe53-55-11 and the wild-type Fielder plants contained more cellulose in their cell walls, whereas the edited plants Xe53-17-2, Xe53-25-9, Xe53-8-2, and Xe53-13-4 contained more lignin in their cell walls (Fig. 4). Therefore, the TaAQ-edited plants Xe53-17-2 and Xe53-25-9 and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4 were more brittle than the wild-type plants, the TaDq-edited plants Xe53-38-4 and Xe53-55-11, and the TaAQ-edited plant Xe53-91-7, which only had a 3-bp deletion in the TaAQ sgRNA sequence.
Fig.
4.
Transverse sections of rachis in the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited wheat plants in T1 generation at two weeks after flowering. A–H: Wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively. The transverse sections of rachis were stained with Safranin O/Fast Green.
Next, we analyzed the expression levels of some representative A-class, B-class, C-class, E-class, and the TaTPL genes before the heading stage of the TaAQ- or TaDq-edited plants, which were deduced to influence wheat spike development (Liu et al., 2018; Li et al., 2019; Debernardi et al., 2020). The transcript levels of the AP2-like genes TaAP2L1 and TaAP2L7 were reduced in the edited plants Xe53-17-2, Xe53-25-9, Xe53-38-4, Xe53-55-11, Xe53-8-2, and Xe53-13-4, whereas no significant difference of expression levels was detected forTaAP2L2 between the edited plants and the wild-type Fielder plants (Fig. 5A–C). The expression level of TaTPL was significantly lower in the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4 (Fig. 5D). The expression levels of the A-class genes VRN1 and FUL2 were reduced in the six mutants except for Xe53-91-7, whereas the expression level of FUL3 did not significantly differ in all the mutants compared with the wild-type plants (Fig. 5E–G). In mutants Xe53-17-2, Xe53-25-9, Xe53-8-2, and Xe53-13-4, the expression levels of genes TaPI1, TaAP3, TaSEP3, TaSEP4, TaSEP5, and TaSEP6 were downregulated, whereas the expression levels of genes TaAG1 and TaAG2 were upregulated. The expression level of the TaSEP2 gene was reduced significantly in all the mutants except Xe53-91-7 (Fig. 5). Consequently, the expression levels of TaTPL, AP2-like genes, and A-, B-, C-, and E-class genes among the TaAQ-edited plants (Xe53-17-2 and Xe53-25-9) and the TaAQ and TaDq simultaneously-edited plants (Xe53-8-2 and Xe53-13-4) displayed similar trends. The expression levels of TaTPL, VRN1, TaPI1, TaAP3, TaSEP3, TaSEP4, TaSEP5, and TaSEP6 were slightly downregulated and the expression levels of TaAG1 and TaAG2 were slightly upregulated in the TaDq-edited plants Xe53-38-4 and Xe53-55-11. However, the expression levels of theTaTPL gene, AP2-like genes, and A-, B-, C-, and E-class genes in mutant Xe53-91-7, which had a 3-bp deletion in the TaAQ sgRNA sequence, were consistent with the wild-type Fielder plants (Fig. 5).
Fig.
5.
Expression levels of the AP2-like, TaTPL, A-, B-, C-, and E-class genes in the developing inflorescences of the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited T1 wheat plants. A–P: The relative expression levels of TaAP2L1, TaAP2L2, TaAP2L7, TaTPL, VRN1, FUL2, FUL3, TaPI1, TaAP3, TaAG1, TaAG2, TaSEP2, TaSEP3, TaSEP4, TaSEP5, and TaSEP6 genes, respectively. Samples 1 to 8 were the wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively. All samples were analyzed with three biological replicates. Significance at P < 0.05 andP < 0.01 is indicated by single or double asterisks, respectively.
2.4
Genetic stability of the TaAQ- and TaDq-edited plants in T2 generation
To confirm the genetic stability of the TaAQ- and TaDq-edited plants and the continuous activity of gene SpCas9 in T2 generation, seven types of T1 transgenic plants, Xe53-3-5, Xe53-91-7, Xe53-91-1, Xe53-55-11, Xe53-55-3, Xe53-38-4, and Xe53-38-1, which contained the SpCas9 cassette were tested (Fig. S7). Xe53-91-7 and Xe53-91-1 had a 3-bp deletion at the 3 bp upstream of the PAM sites in the sgRNA sequence of TaAQ; Xe53-55-11, Xe53-55-3, Xe53-38-4, and Xe53-38-1 had a 4-, 4-, 1-, and 1-bp deletion at the 3 bp upstream of the PAM sites in the TaDq sgRNA sequence, respectively; Xe53-3-5 had no detectable edited event, and Xe53-91-7, Xe53-55-11, and Xe53-38-4 were homozygous knockout plants, whereas Xe53-91-1, Xe53-55-3, and Xe53-38-1 were heterozygote knockout plants in T1 generation (Table 2). We tested 28, 25, 30, 28, 25, 30, and 29 T2 progenies derived from T1 plants Xe53-3-5 to Xe53-38-1 by Sanger sequencing, respectively. All the phenotypes of the TaAQ and TaDq knockout mutants in T2 generation were consistent with those in T1 generation; no new mutation type was observed in T2 generation. The segregation ratio (1:2:1) of the biallele mutant, heterozygote mutant, and the wild type in all the mutants was in accordance with the Mendelian genetics (Table 2). All the aforementioned results indicate that genome editing in wheat occurs only during the tissue culture steps of the experiments. Moreover, despite the presence of the SpCas9 cassette in the edited plants, no new phenotype was produced in this generation.
Table
2.
Inheritance of different knockout types for TaAQ and TaDq genes in T1 and T2 generations.
2.5
Detection of off-target mutation in T1-edited plants
To verify specificity of the CRISPR/SpCas9 system for the TaQ genes in wheat, we evaluated seven potential off-target positions based on the sgRNA sequences of the TaQ genes in wheat reference genome. They had 2–5 mismatched base pairs with the TaQ target sequences (Table S2). The PCR-RE assay and Sanger sequencing using the specific primers for the potential off-targets showed that no mutation occurred at all the seven possible off-target sites in the T1-edited wheat plants. These results indicate that the possible off-target can be neglected, and the editing for the wheat TaQ genes by the CRISPR/SpCas9 system was highly specific.
3.
Discussion
3.1
Knockout of the TaQ genes leading to changes in wheat botanic traits
In wheat, locus Q like other AP2-like genes was one of the targets of miR172. The miR172-overexpression wheat plants and the TaAQ gene loss-of-function wheat mutants exhibited a speltoid-like spike phenotype, increased sclerenchyma cells in glumes, reduced glume angle, and enhanced glume tenacity. Notably, the TaAQ loss-of-function mutants were difficult to be threshed (Zhang et al., 2011; Debernardi et al., 2017; Liu et al., 2018). It was confirmed that the TaAQ/q gene influenced numerous wheat domestication traits (Simons et al., 2006). In the mutants of Chinese Spring, the deletion of TaAQ or its replacement by TaAq affected glume toughness, threshability, rachis fragility, spike length and shape, spike emergence time, and plant height (Zhang et al., 2011).
In the present study, the T0TaQ-edited plants derived from the wheat cultivars Fielder and Jimai 22 showed significant changes in spike architecture (Fig. 1). A few TaQ loss-of-function mutants showed a greater number of sterile florets and a longer spike, shorter awn length, and reduced glume angle (Fig. 1A–E). Owing to the elongated rachis segments, the T0-edited plants from Jimai 22 showed a more spear-shaped spike than the wild-type plants (Fig. 1F–J). In addition, the results on rachis staining showed that the TaQ-edited plants contained more lignin than cellulose in their cell walls, whereas the wild-type Jimai 22 plants contained more cellulose than lignin in the cell walls, which lead to brittle spikes of the TaQ-edited plants (Fig. 2A and B).
Similarly, a previous report found that the brittle rachis trait of the TaQ mutants was affected by the greater accumulation of lignin in cell walls (Jiang et al., 2019). The edited wheat plants obtained in this study had stronger keels and more abundant sclerenchyma in the transverse sections of the lateral glume regions (Fig. 2C–F), supporting the similar results reported in a previous study by overexpressing miR172 in wheat (Debernardi et al., 2017).
3.2
Knockout of the TaQ genes leading to the changes of wheat agronomic traits
It was found in recent studies that the pollens from the TaQ loss-of-function mutants and the Taq plants were smaller, shrunken, and poorly stained with I2-IK than those from the wild-type plants, indicating that pollen viability is likely reduced (Zhang et al., 2020). The TaQ-edited wheat plants generated in this study were similar in terms of spike phenotype to the miR172-overexpression plants or the TaQ loss-of-function mutants and the Taq plants. Hence, we inferred that the malformed pollen must have resulted in the reduction of the seed setting rate.
Of the two phenotypes from the Fielder TaQ-edited plants, seed setting rates were significantly lower to where no seed could be harvested from one of the mutants and prohibited further study of this line despite that many florets per spikelet were produced (Fig. 1). In a previous study also, no seed was harvested in two of 14 transgenic wheat plants overexpressing miR172 (Debernardi et al., 2020).
The spike shape of the TaAQ-edited wheat plants Xe53-17-2 and Xe53-25-9 shifted to a speltoid-like phenotype, and their spikelet, seeds per spikelet, and total seed numbers were reduced. Zhang et al. (2011) found that TaDq also had an important function in controlling spike-related traits, including spike shape, spikelet number, and glume toughness, where TaAQ was absent; the wheat lines with a double deletion of TaAQ and TaDq showed speltoid-shaped spikes, fewer spikelets per spike, and lower fertility than the parental TaAQ or TaDq deletion lines, which consequently resulted in low grain yield.
However, in our present study, wheat spike shape, spikelets per spike, seeds per spikelet, and plant height were not significantly different between TaAQ and TaDq simultaneously-edited and TaAQ-edited plants (Fig. 3 and S6). Reportedly, the wheat mutation at the miR172-binding site in TaDq might cause spike compactness and reduced plant height (Zhao et al., 2018). The spike shape of the TaDq-edited wheat plants was similar to the wild-type plants, except a slight reduction in plant height (Fig. S6). In addition, the rachis of the TaAQ-edited and the TaAQ and TaDq simultaneously-edited plants was more brittle than that of the TaDq-edited and the wild-type plants (Fig. 4).
3.3
Expression regulation of floral organ development-related genes by TaQ genes in wheat
It was well known that the gene-specific transcriptional activators and repressors together with common coactivators and corepressors regulated gene expression. TOPLESS (TPL) and TPL-like proteins were transcriptional corepressors in plants (Causier et al., 2012). In maize and rice, TPL-like proteins played a key function in controlling inflorescence architecture (Yoshida et al., 2012). Liu et al. (2018) found that the Q protein was regulated by tae-miR172 and transcriptional corepressor TaTPL and controlled wheat spike architecture. We observed that the expression level of TaTPL was downregulated in the TaAQ-edited, TaDq-edited, and TaAQ and TaDq simultaneously-edited wheat plants, except for the edited plant Xe53-91-7 having a 3-bp deletion in the TaAQ sgRNA sequence (Fig. 5D). The downregulation expression of TaTPL was more obvious in the TaAQ and TaDq simultaneously-edited plants than that in the TaAQ- or TaDq-edited plants, which confirms that the expression level of TaTPL is regulated by TaQ.
The AP1-like and AP2-like genes determined the identity of sepals and petals in Arabidopsis (Whipple et al., 2004). Wheat AP1-like genes VRN1, FUL2, and FUL3, belonging to the MIKC-type MADS-box proteins, played a major role in controlling generation of the primary and secondary branches from the inflorescence meristem and determining the lateral spikelet meristems and subtending bracts (Li et al., 2019). Debernardi et al. (2020) found that the expression levels of the A-class genes VRN1, FUL2, and FUL3 in wheat had no significant difference in the wild-type plants and ap2l2, ap2l5, and ap2l2ap2l5 mutants. However, in the present study, the expression levels of VRN1 and FUL2 were downregulated in the TaAQ-edited, TaDq-edited, and TaAQ and TaDq simultaneously-edited wheat plants, whereas the expression level of FUL3 did not change across all the mutants (Fig. 5E–G).
In rice and maize, the B-class genes were found to control lodicule, petal, and stamen development (Whipple et al., 2004). In wheat, the transcript levels of the B-class orthologue genes TaPI1 and TaAP3 were significantly reduced in the TaAQ-edited and the TaAQ and TaDq simultaneously-edited plants (Fig. 5H and I), which were consistent with the expression profiles in ap2l5 and ap2l2 ap2l5 mutants (Debernardi et al., 2020).
The expression levels of the C-class genes AG1 and AG2 were repressed by the AP2-like genes (Drews et al., 1991; Debernardi et al., 2020). The homologues of the C-class genes in rice partly regulated the production of stamens and carpels (Yamaguchi et al., 2006). We found that the expression levels of TaAG1 and TaAG2 genes were significantly upregulated in the TaAQ-edited and the TaAQ and TaDq simultaneously-edited plants (Fig. 5J and K).
The SEP-like genes (E-class) had two subfamilies SEP3 and LOFSEP (Malcomber and Kellogg, 2005). In rice, the SEP3 and SEP4 genes in the SEP3 subfamily controlled the characteristics of lodicules, stamens, and carpels (Cui et al., 2010), whereas the SEP2, SEP5, and SEP6 genes in the LOFSEP subfamily regulated specification of spikelets and floral organs (Cui et al., 2010; Wu et al., 2018). The expression levels of TaSEP2, TaSEP3, and TaSEP4 genes were downregulated in the wheat ap2l5 and ap2l5ap2l2 mutants, whereas the expression levels of TaSEP5 and TaSEP6 had no significant difference in the wild-type plants and the ap2l2, ap2l5, and ap2l5ap2l2 mutants (Debernardi et al., 2020). However, the expression levels of all the E-class genes were downregulated in the TaAQ-edited and TaAQ and TaDq simultaneously-edited wheat plants in our present research (Fig. 5L–P).
All the aforementioned results indicate that the expression levels of the A-, B-, C-, and E-class genes, especially that of the TaTPL gene, were closely related to the expression of the TaQ gene. , the AP1-like genes VRN1 and FUL2 and the LOFSEP-subfamily genes SEP2, SEP5, and SEP6 were downregulated in the TaAQ-edited and the TaAQ and TaDq simultaneously-edited wheat plants (Fig. 5). Previous investigations revealed that the spike shape of wheat was controlled by the Q protein, which was regulated by the gene TaTPL (Liu et al., 2018). In addition, the number of spikelets and florets per spike was influenced by VRN1, FUL2, and the LOFSEP-subfamily genes (Wu et al., 2018; Li et al., 2019). Considering that TaQ is a regulatory gene, we postulated that VRN1, FUL2, and the LOFSEP-subfamily genes might be similar to TaTPL gene, regulated the expression of the Q protein, and controlled the number of spikelets and florets per spike in wheat.
3.4
Possible regulatory network of wheat botanic and agronomic traits by TaQ-associated genes and miR172
Combining our results from wheat and previous reports from other plants such as Arabidopsis, rice, and maize (Whipple et al., 2004; Malcomber and Kellogg, 2005; Liu et al., 2018; Debernardi et al., 2020), we conceived a regulatory network of various botanic and agronomic traits in wheat including plant height, spike shape, and floral organs by the genes in the AP2-like family (Fig. 6). We thought in wheat that the A-class genes determine the identity of lemma and palea; the A-, B-, and SEP3-subfamily genes control the characteristics of lodicules; the B-, C-, and SEP3-subfamily genes regulate the specification of stamens; the C- and SEP3-subfamily genes control carpel development; TaAQ, VRN1, FUL2, and the LOFSEP-subfamily genes control the number of spikelets and florets per spike; TaAQ influences spike shape, flowering time, glume angle, glume tenacity, rachis fragility, and seed threshability; and TaDq influences plant height. Because miR172 regulated the expression of the AP2-like genes, it also regulated spike characteristics, plant height, and floral organ identity by targeting TaQ (Fig. 6). However, the underlying molecular mechanism connecting VRN1, FUL2, LOFSEP-subfamily, and TaAQ genes needs to be further studied.
Fig.
6.
Regulatory network of plant height, spike shape, and floral organs in wheat by the genes in the AP2-like family. The A-class genes determine the identity of lemma and palea. The A-class, B-class, and SEP3-subfamily genes control the characteristics of lodicule. The B-class, C-class, andSEP3-subfamily genes regulate the specification of stamens. The C-class and SEP3-subfamily genes control carpel development. TaAQ, VRN1, FUL2, and the LOFSEP-subfamily genes control the number of spikelets and florets per spike. TaAQ influences spike shape, flowering time, glume angle, glume tenacity, rachis fragility, and seed threshability. TaDq influences plant height. miR172 also regulates spike characteristics, plant height, and floral organ identity by targeting the TaQ gene.
3.5
Happening of conjoined plants in wheat transformation and genome editing
Traditionally, a single T0 transgenic wheat plant was thought to be one transgenic event. We also regarded the transgenic plantlets originated from the same embryo as one transgenic event in a crop. However, in this study, different phenotypes were observed in different tillers of a single transgenic plant containing the SpCas9 expression cassette (Fig. 7). Twelve spikes from three T0 transgenic plants obtained from Jimai 22 were analyzed by the PCR-RE assay, and only four of those spikes exhibited the TaAQ-edited phenotype, and the other eight spikes did not show the specific phenotype (Fig. S2C and D). Therefore, different spikes in a single transgenic plant might be independent transgenic events or differentially edited types (e.g., the biallelic or heterozygous mutants with the TaAQ- or TaDq-edited event). We speculated that different transformants were closely grown together during embryogenesis in the selection process of in vitro culture. Thus, we must have generated conjoined plants, similar to the phenomenon of conjoined babies, from which a negative transformant could be survived by virtue of the roots of the positive transformant.
Fig.
7.
Spike-shape phenotypes of the T0 transgenic wheat plants from Jimai 22. A: Spike phenotype of one transgenic plant in T0 generation. B: Enlargement of the spike in (A) in red box, and the red arrow represented the speltoid-like shape. C: PCR products of the SpCas9 gene in T0 transgenic plants. Lanes 1–6: amplification of the SpCas9 gene in T0 transgenic plants. Lane 7: amplification of the SpCas9 gene in the wild-type Jimai 22. Lane 8: amplification of the SpCas9 gene in vector p110-SpCas9-TaQ. M: 2-kb DNA ladder. PCR: polymerase chain reaction.
In addition, we found that the base-pair deletions in the TaQ alleles were the dominant type of mutation in the T0-edited wheat plants. Deletions of 1, 2, 3, 4, 5, or 52 bp at the 3 bp upstream of the PAM sites in the sgRNA sequence were identified in the TaAQ-edited wheat plants, and deletions of 1, 2, 4, or 5 bp at the 3 bp upstream of the PAM sites were identified in the TaDq-edited wheat plants (Fig. S3). All the knockout edited types of TaAQ and TaDq in T2 generation were consistent with those in T1 generation, and no new mutation type was detected in T2 generation despite the presence of SpCas9 in the seven selected lines (Table 2, Fig. S7). The consistency of the mutation types from generation to generation was congruent with previous reports (Zhu et al., 2016; Howells et al., 2018; Liu et al., 2020b). However, one study reported that new edited mutation types did occur, and the editing efficiencies were increased in the next few generations (Zhang et al., 2019a). Considering these contrasting results, we presented two possible explanations: 1) some T0 transgenic plants might be conjoined with nontransgenic plants; 2) some edited plants might be escaped for detection in T0 generation. Moreover, target site designing and promoter choosing for sgRNA are also potential sources of the conflicting results.
4.
Materials and methods
4.1
Plant materials and growth conditions
A spring wheat variety, Fielder, and a winter wheat variety, Jimai 22, were obtained from the National Crop Germplasm Preserve Center at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. Jimai 22 seedlings were vernalized in a refrigerator at 4 °C for a month before transplanting. Fielder seeds and the vernalized Jimai 22 seedlings were grown in pots (20 cm × 30 cm) filled with peat moss (King Root, Beijing, China) mixed with patterned release fertilizer (Osmocote Exact, Germany) and maintained in a growth chamber at 24 °C/16 h in light (300 μmol m−2 s−1 for light density) and 18 °C/8 h in dark conditions, both at 45% humidity. Wheat plants were watered once a week. Pests were controlled with colored sticky cards (Oukeqi Instruments Ltd., Zhengzhou, China). Immature wheat grains were collected from the wheat plants 15 days post anthesis (DPA) for genetic transformation (Wang et al., 2017).
4.2
Vector construction for gene editing
Plasmid pWMB110 was previously constructed by our laboratory (Fig. S1), and the vector containing SpCas9 was kindly provided by Prof. Yaoguang Liu at Southern China Agricultural University, Guangzhou, China. The SpCas9 gene was cloned onto the vector pWMB110 under the control of the maize ubiquitin (ubi) promoter to generate plasmid pWMB110-SpCas9. The promoter TaU3 was amplified from Fielder to control the target sgRNA of the TaQ gene. Based on a previous report, TaBq on chromosome 5BL was a pseudogene, and its sequence was corresponding to the sequence of TaAQ; but in TaBq, a portion of 531 bp, including a 354-bp open reading frame (ORF) between exons 1 and 5, was missing, and the sequence corresponding to intron 7 of TaAQ was not spliced out (Zhang et al., 2011). Therefore, we designed one sgRNA sequence simultaneously targeting the third exon of the two TaQ homologous genes on chromosomes 5AL (TraesCS5A02G473800) and 5DL (TraesCS5D02G486600). The target sequences are shown in Fig. S2B. In accordance with the methods described by Ma et al. (2015), the sgRNA was fused with promoter TaU3 and inserted into the CRISPR/SpCas9 plant transformation vector and named ‘p110-SpCas9-TaQ’ (Fig. S2A). p110-SpCas9-TaQ was further verified by sequencing and introduced into Agrobacterium strain C58C1 by triparental mating (Ditta et al., 1980).
4.3
Agrobacterium-mediated wheat transformation
Immature wheat grains collected from the growth chamber were surface sterilized with 75% ethanol for 1 min, then submerged in 15% sodium hypochlorite (NaClO) for 10 min, and finally rinsed four times with sterile water under aseptic conditions. We used a proprietary method for Agrobacterium-mediated transformation of wheat developed by the Japan Tobacco Company (Tokyo, Japan) (Ishida et al., 2015) with minor modifications (Wang et al., 2017). Transgenic wheat plants were maintained in the growth chamber under the aforementioned growing conditions.
4.4
Detection and identification of edited plants
Genomic DNA was isolated from putative T0 transgenic wheat plants. Specific primers for the bar and SpCas9 genes were designed to screen and identify positive transgenic plants (Table S1). We conducted the PCR amplification using the One Step Mouse Genotyping Kit (Vazyme Biotech Co., Ltd. Nanjing, China) with a Bio-Rad C1000 thermal cycler (Bio-Rad, USA) at 95°C for 5 min, followed by 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s and 5 min for final extension at 72°C. The PCR-RE was used to detect the edited plants. The target regions of genes TaAQ and TaDq were amplified by their specific primers using the aforementioned method (Table S1), and then, the PCR products were digested with the restriction enzyme Not I in a 20-μL reaction system containing 0.2 U Not I and 10-μL PCR product for 3 h at 37°C. The digested and undigested products were separated by electrophoresis on a 2.0% agarose gel and visualized using a GelDoc XR System (Bio-Rad, USA).
The PCR products of the edited plants were subcloned into vector pMD18-T (TaKaRa, Dalian, China) and directly Sanger sequenced. For each mutant sample, at least five positive colonies were randomly selected and sequenced. The mutation types were identified by aligning their sequences with the reference sequences TraesCS5A02G473800 for TaAQ and TraesCS5D02G486600 for TaDq.
4.5
RNA extraction and gene expression analysis by quantitative real-time PCR assay
Samples were collected from the developing inflorescence tissues of the edited wheat plants and ground to fine powder in liquid nitrogen. RNA was extracted from the powder samples using the Trizol (Thermo Fisher Scientific, Boston, USA) reagent following the manufacturer's instructions. Then, cDNA was synthesized from 1 μg of total RNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co., Ltd) at 37°C for 15 min and 85°C for 5 s. Quantitative real-time PCR (qRT-PCR) was performed to analyze the expression levels of the TaQ gene, AP2-like genes (TaAP2L1, TaAP2L2, and TaAP2L7), a Q protein interactional gene (TOPLESS [TaTPL]),APETALA1 (AP1)-like (A-class) genes (VERNALIZATION 1 [VRN1],FRUITFULL2 [FUL2], andFRUITFULL3 [FUL3]), B-class genes (PISTILLATA1 [TaPI1] andAPETALA3 [TaAP3]), C-class genes (AGAMOUS [TaAG1 and TaAG2]), and E-class genes (SEPALLATA [TaSEP2, TaSEP3, TaSEP4, TaSEP5, and TaSEP6]). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China) in a Applied Biosystem 7500 (Thermo Fisher Scientific, Boston, USA) using a thermal cycling program at 95°C for 5 min, followed by 40 cycles of amplification (95°C for 5 s, 60°C for 15 s, 72°C for 15 s).TaADP was used as an endogenous control to normalize quantified mRNA data. All the primers used for the qRT-PCR analysis are listed in Table S1. All samples were analyzed with three biological replicates.
4.6
Tissue staining analysis
Green glumes and rachises were collected 15 DPA from different TaQ-edited wheat plants and fixed in FAA solution (70% alcohol: 37% formaldehyde: acetic acid, 18:1:1, v/v/v) for at least 24 h. The samples were then embedded in paraffin and cut into 6-mm sections using a tissue slicer to investigate anatomical changes of glumes and contents of lignin and cellulose in cell walls. The glume sections were stained with toluidine blue (Solarbio, Beijing, China) for 30 s, and the rachis sections were stained with Safranin O/Fast Green (Solarbio, Beijing, China) for 60 s. Lignin-rich or cellulose-rich cell walls were preferentially stained by Safranin O or Fast Green, respectively. The treated samples were observed and photographed using an Olympus BX-51 microscope with a Photometric SenSys Olympus DP70 CCD camera (Olympus Corporation, Tokyo, Japan).
4.7
Off-target analysis
Potential off-target sites in the CRISPR/SpCas9 system for the TaQ gene were identified using the BLAST tool for alignment in the wheat genome sequence (EnsemblPlants: http://plants.ensembl.org/Triticum_aestivum/Info/Index). Potential off-target sequences were identified by having fewer than 5-bp mismatches when compared with theTaQ target sequences (Table S2). Specific primers for the potential off-targets were designed (Table S2), and PCR-RE assay and Sanger sequencing were used to analyze the mutants.
4.8
Statistical analysis
The SPSS 19.0 software (SPSS, Chicago, USA) package was used for statistical analysis. Statistical comparisons of multiple sets of data were carried out using Duncan's multiple range test. The histogram was constructed using WPS Office software (Kingsoft, Beijing, China).
CRediT authorship contribution statement
Xingguo Ye and Xinwu Pei conceived the research. Ke Wang and Xingguo Ye designed the experiments. Huiyun Liu and Qiang Gong constructed expression vectors. Huiyun Liu, Ke Wang, and Lipu Du carried out wheat transformation. Huiyun Liu and Huali Tang performed expression profiling and histological observation. Huiyun Liu, Xingguo Ye, and Xinwu Pei performed phenotypic investigation. Huiyun Liu, Ke Wang, and Xingguo Ye drafted and revised the manuscript.
Acknowledgments
We thank Prof. Yaoguang Liu at Southern China Agricultural University for providing the CRISPR/Cas9 vector and Prof. Xueyong Zhang at Institute of Crop Sciences, CAAS, for opening the Olympus BX-51 microscope used in this study. This research was financially supported in part by grants from the Ministry of Agriculture and Rural Affairs of China (2016ZX08009001 and 2016ZX08010004), the Science and Technology Department of Ningxia China (2019BBF02020), and the Chinese Academy of Agricultural Sciences (2060302-2-19).
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Figure 1. Spike morphology of the TaQ-edited wheat plants in T0 generation at two weeks after flowering. A [1],B, and C: The wild-type Fielder. A [2],A [3],D and E: The TaQ-edited plants derived from Fielder. F [1],G, and H: The wild-type Jimai 22. F [2],I, and J: The TaQ-edited plants derived from Jimai 22.
Figure 2. Transverse sections of the rachis and the second glume from central spikelets of the T0 edited wheat plants at two weeks after flowering. A and B: The transverse sections of rachis were stained with Safranin O/Fast Green. C–F: The transverse sections of glume were stained with toluidine blue. A, C, and E: The wild-type Jimai 22. B, D, and F: The TaQ-edited Jimai 22 plants.
Figure 3. Spike morphology of the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited wheat plants in T1 generation at two weeks after flowering. Samples 1 to 8 were the wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively.
Figure 4. Transverse sections of rachis in the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited wheat plants in T1 generation at two weeks after flowering. A–H: Wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively. The transverse sections of rachis were stained with Safranin O/Fast Green.
Figure 5. Expression levels of the AP2-like, TaTPL, A-, B-, C-, and E-class genes in the developing inflorescences of the TaAQ-, TaDq-, and TaAQ and TaDq simultaneously-edited T1 wheat plants. A–P: The relative expression levels of TaAP2L1, TaAP2L2, TaAP2L7, TaTPL, VRN1, FUL2, FUL3, TaPI1, TaAP3, TaAG1, TaAG2, TaSEP2, TaSEP3, TaSEP4, TaSEP5, and TaSEP6 genes, respectively. Samples 1 to 8 were the wild-type Fielder; the TaAQ-edited plants Xe53-91-7, Xe53-17-2, and Xe53-25-9; the TaDq-edited plants Xe53-38-4 and Xe53-55-11; and the TaAQ and TaDq simultaneously-edited plants Xe53-8-2 and Xe53-13-4, respectively. All samples were analyzed with three biological replicates. Significance at P < 0.05 andP < 0.01 is indicated by single or double asterisks, respectively.
Figure 6. Regulatory network of plant height, spike shape, and floral organs in wheat by the genes in the AP2-like family. The A-class genes determine the identity of lemma and palea. The A-class, B-class, and SEP3-subfamily genes control the characteristics of lodicule. The B-class, C-class, andSEP3-subfamily genes regulate the specification of stamens. The C-class and SEP3-subfamily genes control carpel development. TaAQ, VRN1, FUL2, and the LOFSEP-subfamily genes control the number of spikelets and florets per spike. TaAQ influences spike shape, flowering time, glume angle, glume tenacity, rachis fragility, and seed threshability. TaDq influences plant height. miR172 also regulates spike characteristics, plant height, and floral organ identity by targeting the TaQ gene.
Figure 7. Spike-shape phenotypes of the T0 transgenic wheat plants from Jimai 22. A: Spike phenotype of one transgenic plant in T0 generation. B: Enlargement of the spike in (A) in red box, and the red arrow represented the speltoid-like shape. C: PCR products of the SpCas9 gene in T0 transgenic plants. Lanes 1–6: amplification of the SpCas9 gene in T0 transgenic plants. Lane 7: amplification of the SpCas9 gene in the wild-type Jimai 22. Lane 8: amplification of the SpCas9 gene in vector p110-SpCas9-TaQ. M: 2-kb DNA ladder. PCR: polymerase chain reaction.
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