Creation And Identification Of Mutants In Rice With Ideal Plant Architecture

Rice is one of the important grain crops in the world,and nearly half the population of the world feeds on rice.The yield of rice is always considered to be the key index to assess whether the rice cultivar is of high quality,and the cultivation of the ideal plant architecture is not only an important way to get high-yielding rice,but also a major agronomic trait which decides the lodging resistance.By far,although many genes related to the ideal plant architecture have been identified in many aspects,such as rice stalk,blade,spike shape,panicle number,plant character and root,it is difficult to combine various genes of ideal plant architecture via traditional cross breeding and backcross breeding.In the present study,basing on CRISPR-Cas9 technology-mediated targeted genome editing,we have created mutant materials with single or multiple genes for three genes of regulation of rice plant architecture,OsSPL14,OsTB1 and OsDEP1.Then we identify the plant architecture of mutants,and assess the availability of CRISPR-Cas9 technology-mediated targeted genome editing when it is applied to research the ideal rice plant architecture.It is hoped that this study can provide the molecular tool and typical example for creating the ideal plant architecture and increasing the rice yield.The main research contents and results are as follows:1.We designed two sgRNA targeting sites for the coding sequence of OsDEP1 gene,and we constructed the CRISPR-Cas9 expression vector to precisely modify target gene.Then we screened resistant plants after Agrobacterium-mediated rice transformation,and identified the mutant plants on the target site.The experimental results are as follows: the mutagenesis frequency at OsDEP1-sgRNA1 target site was 75%,and bi-allele mutagenesis frequency was 66.7%;the mutagenesis frequency at OsDEP1-sgRNA2 target site was 58.3%,and bi-allele mutagenesis frequency was 25%.The plant pTX202-03 was the mutant including 1bp deletion at OsDEP1-sgRNA1 target site,and twenty plants of T1 generation that we identified were homozygous mutants,which were the same as its parents.Besides,the whole modified materials of T0 and T1 generation showed a representative dwarf character.The results indicated that the genotype and phenotype were modified by CRISPR-Cas9 on OsDEP1.2.We constructed three expression vectors for modifying the coding sequence of OsSPL14 gene at different sgRNA target site,and the mutagenesis frequency was as follows: 93.9%(bi-allele mutagenesis frequency was 53.1%,OsSPL14 sgRNA1),63.6%(bi-allele mutagenesis frequency was 18.2%,OsSPL14-sgRNA2),63.6%(bi-allele mutagenesis frequency was 36.4%,OsSPL14-sgRNA4),respectively.3.We constructed four expression vectors for modifying the coding sequence of OsTB1 gene at different sgRNA target site,and the mutagenesis frequency was as follows: 33.3%(bi-allele mutagenesis frequency was 22.2%,OsTB1-sgRNA1),0.0%(OsTB1-sgRNA2),15.4%(bi-allele mutagenesis frequency was 7.7%,OsTB1-sgRNA3),15.8%(biallele mutagenesis frequency was 0.0%,OsTB1-sgRNA4),respectively.4.We constructed five expression vectors for modifying the coding sequence of OsSPL14 and OsTB1 gene at double target site,while,we failed to obtain the mutant plants with double site knock-out from pZDW026(OsSPL14-sgRNA2+OsTB1-sgRNA3),pZDW027(OsSPL14-sgRNA4+OsTB1-sgRNA3)and pZDW028(OsSPL14-sgRNA1+OsTB1-sgRNA3).Fortunately,the mutagenesis frequencies with double site knock-out were 50% and 11.8% from p ZDW052(OsSPL14-sgRNA1+Os TB1-sgRNA1)and pZDW053(OsSPL14-sgRNA1+OsTB1-sgRNA4),respectively.