Development Of Genetic Engineered Thermo-sensitive Genetic Male Sterile Rice Lines Using The CRISPR/Cas9 System

As one of the world?s most importa nt crops,Asian cultivated rice?Oryza sativa L.?has attract much attention.Utilization of heterosis is a major way to increasethe yieldof rice.The three-line hybridsystems and the two-line hybrid systemshave been developed for this purpose.With the application of thethermo-sensitive genetic male sterile?TGMS?lines,the two-line systems are more efficient for utilizing heterosis.Annong S-1is the first found indica rice TGMS line in the world.Its thermos-sensitivemale sterility related gene,tms5,has been mapped and cloned.TheC RISPR/Cas9 system has quickly evolved since2013,and has been widely used for targeted genome mutagenesis in crops.In this study,various rice varieties were genetically engineered by using the CRISPR/Cas9 system,thusTGMS rice lines of various varietieswere developed.The main results are as follows:1.T₀ transgenic plants of fifteen rice varieties,namely C10,H12B,GA114,M15,T110,W55,P14,NYZ1,NYZ2,NJZ,NGZ,NH2,YXZ,JNSM and NXRZ,were examined for targeted mutagenesis in the thermos-sensitivemale sterility related gene TMS5.Phenotypes of these mutants were confirmed and seeds wereindividually harvested.T₁transgenic plants of six varieties,namely C10,H12B,GA114,T110,W55 and P14,werescreened for??transgene-clean??mutants via PC R and hygromycin resistance test.These mutants were treated with fertility inducing temperature and seeds were individually harvested.2.Mutant plants ofM15,T110 and P14 are tested for the sterility inducing temperature by heat treatment of gradient-temperature.I₂-KI dyeing test of pollen shows the sterility inducing temperatures of M15,T110 and P14 mutant plants are all between 24?and 28?.3.Whole genome sequencings of ZS97B,TFB and YJSM??transgene-clean??TGMS T₂ plants were carried out to evaluate the potential off-target effect of the CRISPR/Cas9system.No off-target effects were detected on 18 putative off-target sites.4.Progenies of??transgene-clean??plants are tested for inheritance characters of targeted mutagenesis.In the cases of Line GA114-23 and Line T110-9,the mutations in TMS5 are stably passed to the next generation.As for Line C10-1 and Line W55-5,inversion between the two CRISPR/Cas9-system targets,which is a new genotype,was detected among the T₂ and T₃ generation,respectively.For biallelic lines,namely Line H12B-16 and GA114-22,targeted mutagenesis are stably passed to the next generation and segregation in T₂ generations conform to classic Mendelian law.5.Pollen of NYZ1 genetically engineered TGMS lines?T₀ generation?of different targeted mutagenesis typesare obtained on the same day and tested for sterility through I₂-KI dyeing.No difference is detectedin this study.According to the above results,preliminarily identified??transgene-clean??genetically engineered TGMS rice lines of various varieties were developed and basic characteristics of TGMS rice lines were analysed.