Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system

doi:10.1111/pbi.13391

. 2020 Nov;18(11):2164-2166.

doi: 10.1111/pbi.13391. Epub 2020 May 13.

Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system

Lichun Huang¹, Qianfeng Li^{1

2}, Changquan Zhang^{1

2}, Rui Chu¹, Zhengwen Gu¹, Hongyan Tan¹, Dongsheng Zhao^{1

2}, Xiaolei Fan^{1

2}, Qiaoquan Liu^{1

2}

Affiliations

¹ Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University, Yangzhou, China.
² Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou, China.

PMID: 32339389
PMCID: PMC7589223
DOI: 10.1111/pbi.13391

Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system

Lichun Huang et al. Plant Biotechnol J. 2020 Nov.

. 2020 Nov;18(11):2164-2166.

doi: 10.1111/pbi.13391. Epub 2020 May 13.

Authors

Lichun Huang¹, Qianfeng Li^{1

2}, Changquan Zhang^{1

2}, Rui Chu¹, Zhengwen Gu¹, Hongyan Tan¹, Dongsheng Zhao^{1

2}, Xiaolei Fan^{1

2}, Qiaoquan Liu^{1

2}

Affiliations

¹ Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University, Yangzhou, China.
² Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou, China.

PMID: 32339389
PMCID: PMC7589223
DOI: 10.1111/pbi.13391

No abstract available

Keywords: Oryza sativa L.; Wx gene; amylose content; core promoter; eating and cooking quality; genome editing.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

**Figure 1**
CRISPR/Cas9 system editing of the Wx promoter to create novel Wx alleles to fine‐tune expression and amylose content. (a) Schematic diagram of predicted *cis*‐acting elements and target sites (S1–S7) for editing on the promoter of *Wx^b* locus. The locations of S1‐S7 are shown in panel (b). (b) The amylose content (AC) in mature grains of T₁ homozygous edited lines in Lingshui (winter). ‘+’, mutation. (c) Alteration of Wx sequences in six S7‐edited lines and their frequencies among the 16 mutated T₀ individuals. (d) Morphology of milled rice from the edited line *Wx^b‐d8* and wild‐type *Wx^b*(WT). (e) The relative expression levels of Wx in developing endosperm. The locations of primers in brackets are arrowed in panel (a). (f) *In vitro* transcriptional activity of Wx promoter (−1463 bp to +1299 bp) from *Wx^b* and its edited alleles in tobacco using dual‐luciferase reporter assay. Vector, pGreenII 0800‐LUC. (g and h) AC and GBSSI accumulation of homozygous S7‐edited lines. GBSSI and HSP, antibodies recognizing GBSSI and the input control heat shock protein (HSP). (i) Comparison of rapid viscosity analyser spectra of rice flours from plants in Lingshui (winter). (j and k) AC in mature grains and mature Wx mRNA levels in developing seeds under different temperature treatments. *Wx^b‐C/A*, *Wx^b‐A/C*, *Wx^b‐i1*, *Wx^b‐d2*, *Wx^b‐d8* and *Wx^b‐d15* indicate the edited lines, and *Wx^mp* (10.57% of AC) and wx (glutinous) are near‐isogenic lines carrying corresponding Wx alleles under the Nipponbare (*Wx^b*) background. At least three plants of each line were sampled for each measurement. Error bars are means ± SD (n = 3), * and ** indicate significant differences at P < 0.05 and P < 0.01 levels, respectively, based on one‐way ANOVA.s

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References

1. Cai, X.L. , Wang, Z.Y. , Xing, Y.Y. , Zhang, J.L. and Hong, M.M. (1998) Aberrant splicing of intron 1 leads to the heterogeneous 5′ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J. 14, 459–465. - PubMed
1. Chen, K.L. , Wang, Y.P. , Zhang, R. , Zhang, H.W. and Gao, C.X. (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697. - PubMed
1. Haberle, V. and Stark, A. (2018) Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 19, 621–637. - PMC - PubMed
1. Lau, W.C.P. , Latif, M.A. , Rafii, M.Y. , Ismail, M.R. and Puteh, A. (2016) Advances to improve the eating and cooking qualities of rice by marker‐assisted breeding. Crit. Rev. Biotechnol. 36, 87–98. - PubMed
1. Li, C.Y. , Li, W. , Zhou, Z.H. , Chen, H. , Xie, C.H. and Lin, Y.J. (2020) A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene‐free bacterial blight‐resistant rice. Plant Biotechnol. J. 18, 313–315. - PMC - PubMed

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[1] Cai, X.L. , Wang, Z.Y. , Xing, Y.Y. , Zhang, J.L. and Hong, M.M. (1998) Aberrant splicing of intron 1 leads to the heterogeneous 5′ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J. 14, 459–465. - PubMed

[2] Cai, X.L. , Wang, Z.Y. , Xing, Y.Y. , Zhang, J.L. and Hong, M.M. (1998) Aberrant splicing of intron 1 leads to the heterogeneous 5′ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J. 14, 459–465. - PubMed

[3] Chen, K.L. , Wang, Y.P. , Zhang, R. , Zhang, H.W. and Gao, C.X. (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697. - PubMed

[4] Chen, K.L. , Wang, Y.P. , Zhang, R. , Zhang, H.W. and Gao, C.X. (2019) CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697. - PubMed

[5] Haberle, V. and Stark, A. (2018) Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 19, 621–637. - PMC - PubMed

[6] Haberle, V. and Stark, A. (2018) Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 19, 621–637. - PMC - PubMed

[7] Lau, W.C.P. , Latif, M.A. , Rafii, M.Y. , Ismail, M.R. and Puteh, A. (2016) Advances to improve the eating and cooking qualities of rice by marker‐assisted breeding. Crit. Rev. Biotechnol. 36, 87–98. - PubMed

[8] Lau, W.C.P. , Latif, M.A. , Rafii, M.Y. , Ismail, M.R. and Puteh, A. (2016) Advances to improve the eating and cooking qualities of rice by marker‐assisted breeding. Crit. Rev. Biotechnol. 36, 87–98. - PubMed

[9] Li, C.Y. , Li, W. , Zhou, Z.H. , Chen, H. , Xie, C.H. and Lin, Y.J. (2020) A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene‐free bacterial blight‐resistant rice. Plant Biotechnol. J. 18, 313–315. - PMC - PubMed

[10] Li, C.Y. , Li, W. , Zhou, Z.H. , Chen, H. , Xie, C.H. and Lin, Y.J. (2020) A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene‐free bacterial blight‐resistant rice. Plant Biotechnol. J. 18, 313–315. - PMC - PubMed

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Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system

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Creating novel Wx alleles with fine-tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system

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