Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (14): 2631-2641.doi: 10.3864/j.issn.0578-1752.2018.14.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Using CRISPR/Cas9-Mediated Targeted Mutagenesis of qSH1 Reduces the Seed Shattering in Rice

SHENG XiaBing1,2, TAN YanNing2, SUN ZhiZhong1,2, YU Dong1,2, WANG XueFeng1, YUAN GuiLong2, YUAN DingYang2, DUAN MeiJuan1   

  1. 1College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128; 2State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Centre, Changsha 410125
  • Received:2018-03-05 Online:2018-07-16 Published:2018-07-16

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Abstract: 【Objective】Seed shattering is not good for seeds harvest，nor suitable for mechanized production of rice. In this study, using the latest molecular breeding method-CRISPR/Cas9 system to site-directed edit of the rice seed shattering gene qSH1 and evaluated the improvement effect, in order to lay materials foundation and explore new method for creating stable-yielding rice germplasm suitable for mechanized seed production. 【Method】The sgRNA was designed based on the principle of CRISPR/Cas9 technology. After excluding non-specific target sites by analysis of sequence alignment in the rice genome database, the target sites of qSH1-T1 and qSH1-T5 were selected. The oligonucleotides of sgRNA were chemically synthesized, and then inserted into plasmid pYLgRNA-U3, pYLgRNA-U6a for constructing the U3-qSH1T5-gRNA and U6a-qSH1T1-gRNA expression-boxes respectively. And the pYLCRISPR/cas9-qsh1-t51 expression vector was constructed by linking expression-boxes to plasmid pYLCRISPR/Cas9. The vector of pYLCRISPR/cas9-qsh1-t51 was introduced into the callus induced from mature seeds of an indica rice variety HR1128 by the Agrobacterium-mediated transformation. At T0 generation, the positive transgenic plants were selected by hygromycin-resistant, and the target site for each plant were detected via a test of PCR combined Sanger-sequencing for confirming whether it was mutated. Besides blasting sgRNA sequence with rice genome in NCBI (https://blast.ncbi.nlm.nih.gov), Gramene (http://ensembl.gramene.org), highly identical sites with more 15 matching bases and NGG site at 3′region were selected to assess off-target efficiency and specificity of sgRNA. By using hpt gene and target site PCR amplification, mark-free qsh1 mutants were selected and qsh1 mutation lines were constructed by qsh1 mutants self-cross, and the level of rice seed shattering, the expression level of qSH1 and amino acid sequence of gene production of qsh1 lines were analyzed.【Result】The pYLCRISPR/cas9-qsh1-t51 expression vector successfully actualized gene-specific editing of qSH1. We obtained 7 mutants in T0 transgenic generation and the mutation type ratios of qSH1-T1, qSH1-T5 were 54.55%, 63.64% respectively. Their genotypes included homozygous, heterozygous, biallelic and chimeric mutations. The mutation types were mainly insertions, deletions and substitution. After analysis of T1 transgenic plants by hpt gene and target site PCR amplification, we obtained 2 mutant types which were homozygous and mark-free, and 2 T2 generation qsh1 mutation lines (namely as 17SZ01, 17SZ02) were constructed further. We examined three putative off-target sites in the rice genome that showed the highly similarity with our target sites. And any off-target mutations at these sites in 46 plants of T0 and T1 generations were not observed, which indicated the sgRNA was highly specificity. Compared with the wild type, the 2 lines showed a significant decreased level of seed shattering (P<0.05). Furthermore, the amino acid sequence were changed of mutation lines, and the qSH1 expression level also decreased significantly (P<0.05) of the 17SZ01 mutation line. 【Conclusion】The strategy of site-directed mutagenesis qSH1 by CRISPR/Cas9 works effective for reducing the seed shattering in rice.

Key words: CRISPR/Cas9, gene editing, rice, qSH1, seed shattering

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