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