Construction And Application Of Seeds Propagation System For Spontaneous Genic Male Sterile Line In The Third Generation Hybrid Rice

Rice is the highest-yield food crop in China,and it also is an important cornerstone for achieving basic self-sufficiency in domestic food and ensuring the absolute safety of grain.The utilization of heterosis has played an important role in continuously improving the yield of rice.At the present stage,the approach of heterosis utilization in rice has undergone the first-generation hybrid rice technology based on cytoplasmic male sterile line(CMS)and the second-generation hybrid rice technology based on environmentally sensitive genic male sterile line(EGMS),and is currently moving towards the third-generation hybrid rice breeding technology based on spontaneous genic male sterile line(SGMS).Spontaneous genic male sterile line has stable and thorough sterility,it is an ideal genetic tool for heterosis utilization and easy to formulate strong dominance hybrid combinations,but the seeds mutiplication problem has always restricted the application of SGMS.In this paper,the control gene of a spontaneous genic male sterility mutant has been cloned,and the seeds propagation system has been constructed using the fertility control genes and sterile mutant,then a targeted breeding technology system of SGMS has been established by gene editing.On these basis,the third generation of hybrid combinations with strong heterosis is breeded through extensive cross with spontaneous genic male sterile line.The main results are shown as follow:1.A non-pollen spontaneous genic male sterility mutant is obtained from the ray mutagenesis offspring of restorer line R299,and its sterility control gene is identified as PTC1.The genetic complementation experiment proves that the wild-type PTC1 gene completely restore the fertility of ptc1 mutant,indicating that PTC1 gene and its sterile mutant are suitable for constructing the seed propagation system of the third-generation hybrid rice sterile line.2.Binary genetic engineering breeding line(BBL)and ternary genetic engineering breeding line(TBL)have been constructed using PTC1 gene and ptc1 sterile mutant combined with endosperm fluorescent marking technology and transgenic pollen killing technology.Both breeding lines can meet the seeds mass propagation of SGMS,when analysis of the effects of different mixed planting ratios of breeding and sterile lines on sterile seeds yield and efficiency,the results show that the optimal mixed planting ratio of BBL and SGMS is 3: 7 and the TBL is 2: 8.However,the overall sterile seeds yield and efficiency of TBL are better than that of BBL.3.Using a series of molecular biology techniques to evaluate the genetic characteristics of BBL and TBL for genetically modified organisms,the results show that the exogenous T-DNA regions can be stably inherited in the rice genome,the insertion sites of the T-DNA region have no adverse effects on BBL and TBL themselves,the foreign genes are stably expressed in the expected tissue of rice,and the expressed foreign protein sequences analysed by bioinformatics have no highly homologous amino acid sequences with known toxic proteins,allergens and resistant nutritional factors.The above results prove that molecular characteristics of BBL and TBL have a lower risk of genetically modified organisms.4.A CRISPR/Cas9 gene editing vector has been constructed based on the PTC1 gene sequence,and a targeted technical system is established for rapidly cultivating spontaneous genic male sterile line.Using this targeted breeding technology,two spontaneous genic male sterile lines of Huazhan-SGMS and TFB-SGMS are obtained,and the stigma exposure rate of both sterile lines exceed 60%,satisfing the requirements of high outcrossing seed setting of sterile lines5.Five high-yielding third-generation hybrid combinations are bred using 299-SGMS and Huazhan-SGMS derived from restorer background,which increase by 5.98%-27.7%over the control hybrid combination Tianyouhuazhan,respectively.This result proves that turning restorer lines into sterile lines is one of the effective ways to breed new sterile lines.Also,the successful of turning restorer into steriler further shows that the third-generation hybrid rice technology can not only break the limitation of the relationship between restorer and maintainer,but also break the limitation of the relationship between restorer and steriler.6.A seamless stacking enzymatic assembly(SSEA)technology is developed to sove the difficult problem of cloning large DNA molecular with complex structures or many restrction sites.The 7.09 kb fertility restoration gene PTC1 is assemble into the vector using SSEA,it lay the foundation for constructing BBL and TBL.7.A method of CDL-PCR for isolating T-DNA flanking sequences is developed,and the T-DNA insertion sites in genetic engineering breeding lines are accurately and efficiently obtained,which provid important molecular date for evaluating the safety of genetically modified organism.