CRISPR/Cas9-mediated restoration of Tamyb10 to create pre-harvest sprouting-resistant red wheat.

Wheat pre-harvest sprouting (PHS) reduces yield and grain quality and occurs in almost every wheat-growing region around the world (Vetch et al., 2019). In general, red-grained wheat varieties are more tolerant to PHS than white-grained varieties (Himi et al., 2011). Moreover, the red pigment of the grain coat contains proanthocyanidins, whose antioxidant activity and free radical scavenging ability have health-promoting properties. Therefore, producing elite red wheat varieties is an important objective in breeding high-yield and high-quality wheat. R2R3-MYB is one of the largest families of transcription factors in plants, which play crucial roles in regulating plant development, metabolism, and stress responses. The R2R3-MYB transcription factor Tamyb10 of hexaploid wheat activates flavonoid biosynthesis genes to specify red grain colour and influences PHS (Himi et al., 2011). In most white wheat varieties, the Tamyb10-A1a, Tamyb10-B1a, and Tamyb10-D1a genes have large insertions or deletions, which disrupt the IRTKAL/IRC motif and regulatory function (Himi et al., 2011). Among the Tamyb10 genes, the Tamyb10-B1a allele has a 19-bp deletion in nearly 88.6% of bread wheat lines; this deletion causes a frameshift in the open-reading frame and disrupts the resulting protein (Dong et al., 2015; Himi et al., 2011). Given that CRISPR/Cas9-induced mutations are typically +1/−1-bp indels at the specific target sites (Zhang et al., 2014, 2016), we can revert the frameshift mutations within the Tamyb10-B1a allele (caused by a 19-bp deletion) to in-frame mutations (18-bp or other multiples of three bases). Here, we first sequenced the Tamyb10-B1a locus in the spring wheat cultivar Fielder and confirmed the 19-bp deletion in the third exon of Tamyb10-B1a in this white wheat variety (Figure 1a; Figure S1). To restore functionality of the Tamyb10-B1a allele, a single gRNA was designed to target the sequences flanking the 19-bp deletion site (Figure 1a; Appendix S1). Then, the pWMB110-SpCas9-sgRNA was constructed and transformed into Fielder via Agrobacterium-mediated transformation (Wang et al., 2022). Fifteen putative transgenic plants were produced, from which five Tamyb10-B1a-edited plants were selected (Figure 1b). Among the T0 edited lines, four plants with a 1-bp insertion upstream of the 19-bp deletion site were identified. These 1-bp insertions restored the reading frame by converting the frameshift mutation in the Tamyb10-B1a allele (19-bp deletion) to in-frame mutation (18-bp deletion; Figure 1b). Furthermore, no mutation events were found across all potential off-target sites (Table S1). To investigate whether the target mutations observed were heritable, we tracked the inheritance of in-frame Tamyb10-B1a variants (NF243-3, NF243-12, and NF243-15). The CRISPR/Cas9-induced mutations in the T0 plants were stably transmitted to the T1 generation without the occurrence of new mutations (Figure 1c; Table S2). Sequence alignment analysis showed that, compared with the wild-type Tamyb10-B1b protein, the protein sequences of the in-frame Tamyb10-B1a variants carried a minor deletion of six amino acids, thereby restoring the intact IRTKAL/IRC motif (Figure 1d; Figure S2). Homozygous mutants with the in-frame Tamyb10-B1a sequence were selected to investigate grain pericarp and PHS resistance phenotypes. As expected, the coloration of the grains harvested from T1 lines harbouring the in-frame Tamyb10-B1a variants changed from white to red (Figure 1e). The accumulation of anthocyanins in these lines was assessed using a spectral imaging chamber. Compared with the wild-type Fielder seeds, the in-frame Tamyb10-B1a lines displayed an elevated anthocyanin index (Figure 1e), suggesting that restoring the Tamyb10-B1a allele caused accumulation of anthocyanidins in wheat grains. Furthermore, the main anthocyanin and proanthocyanidin content in in-frame Tamyb10-B1a seeds was higher than in wild-type seeds (Figure 1f; Figure S3). Our RNA-seq and qRT-PCR data showed that the expression of flavonoid biosynthetic genes increased in the in-frame Tamyb10-B1a lines compared with the wild type (Figure 1g,h; Table S3). The expression levels of Tamyb10-B1a were not affected after editing (Figure S4). These findings confirmed that Tamyb10-B1a allele function was successfully restored. Tamyb10, a gene with pleiotropic effects, is also associated with PHS tolerance of grains (Lang et al., 2021). Our data revealed that seed germination in the in-frame Tamyb10-B1a lines was delayed compared with wild-type threshed seeds (Figure 1i). The germination percentage (GP) of threshed seeds in the in-frame Tamyb10-B1a lines was significantly reduced about 16.18%–32.63% at 5–7 days after imbibition compared with wild-type seeds (Figure 1j). Likewise, the GP of in-frame Tamyb10-B1a mutant seeds also decreased by 24.23% in whole spikes 10 days after imbibition compared with the wild type (Figure 1k,l). The abscisic acid (ABA) content is closely related to PHS tolerance. Therefore, we quantified the levels of endogenous ABA in wild-type and Tamyb10-B1a variant seeds. As shown in Figure 1m, ABA levels were 51.4% higher in seeds in the in-frame Tamyb10-B1a lines compared to the wild type. In addition, both the RNA-seq and qRT-PCR results showed that the expression of ABA biosynthetic genes increased in the in-frame Tamyb10-B1a mutants (Figure 1g,h; Table S3). Furthermore, greater TTC staining was seen in Fielder seeds than in the in-frame Tamyb10-B1a mutant seeds, suggesting that seed coat permeability was reduced in the variants (Figure S5). In summary, we successfully converted a white wheat variety into a red one and improved its PHS tolerance through CRISPR/Cas9-mediated functional restoration of the Tamyb10-B1a allele. It is worth noting that aside from red grains and elevated PHS resistance, the in-frame Tamyb10-B1a variants did not differ from the wild type in other major agronomic traits (Figure S6). In addition, the phenotypes of the edited in-frame Tamyb10-B1a variants were stable after the Cas9 transgene was segregated out (Figure S7). Compared with targeted gene knock-in by homology-directed genome editing, our strategy efficiently recovers gene function without donor DNA and would greatly accelerate the breeding of new red-grained and PHS-resistant wheat varieties with elite agronomic traits. The present study also presents an alternative strategy for crop improvement by editing genes that were lost through domestication via the restoration of reading frames. This work was supported by grants from the National Key Research and Development Program of China (2020YFE0202300), the Guangdong Science and Technology Foundation (2019ZT08N628), The National Natural Science Foundation of China (32022006), and China Postdoctoral Science Foundation (2020M670549). The authors have not declared a conflict of interest. C.-J.Z. and Y.Z. designed the study; Y.Z., Y.L., Y.F., Y.W., P.L., J.X., Y.H., and K.W. performed the experiments; Y.Z., Y.L., S.C., X.Y., F.W., J.G., J.-K.Z., K.W., and C.-J.Z. analysed the data; Y.Z., Y.L., K.W., and C.-J.Z. wrote the article. Figure S1 - S7 Supplementary Figures. Table S1 - S4 Supplementary Tables. Appendix S1 Methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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