Gene Cloning And Combining Ability Analysis Of A Male Sterile Mutant Ptc1-2 In Rice

“three-line”hybrid rice and“two-line”hybrid rice system is well established,which based on cytoplasmic male sterility and photoperiod-and thermo-sensitive genic male-sterile lines to produce Hybrid seeds allows the application of the heterosis.However,photoperiod-and thermo-sensitive male sterility and cytoplasmic male sterility have shortcomings such as unstable fertility and low germplasm utilization in application,which restrict the It is widely used in production.Nucleated male sterility has the advantages of stable fertility,wide available germplasm resources and low seed production costs.Hence,the creation and utilization of nuclear male sterility is the focus of the new generation of rice seed production technology.In this study,a library of indica rice mutants with wide application value was constructed by mutating indica 93-11 seeds with the physical radiation mutagen cobalt60.We screened a male nuclear sterile mutant ptc1-2,And a F₂ isolated population of the mutant was constructed,The target site was obtained by map cloning.The mutation site was then transferred to two line,three line,and conventional rice materials,and their combining ability was detected.Provided material and theoretical basis for future application of nuclear male sterility.The main results were as follows:1.Map cloning of ptc1-2:A F₂ isolated population containing 1362 individual plants was constructed in this study.Fertility segregation statistics show that the mutant/wild type is approximately 1:3,consistent with the regulatory ratio of single-gene recessive inheritance,and therefore,its definitive under single-gene control as a recessive nuclear male sterile mutant.The target mutation was finely localized between marker D3102 and marker D3676 with a physical distance of 573bp using a map base cloning method.Flank sequence separation showed that the mutant had a large segment（about 250kb）deletion on chromosome 9,The fragment contains 19 complete ORFs,one of which is a PTC1 gene.Comparative analysis of 19ORFs and the cloned ptc1 mutant phenotypes,PTC1 shou be the candidate gene for ptc1-2.2.In this study,four nuclear replacement lines of BC₃F₃ generation were obtained,named C815G,Wufeng G,Zhongxiang Huangzheng G and Huazheng G.Phenotypic observations showed that C815G,Wufeng G,Zhongxiang Huangzheng G and Huazheng G were not significantly different in appearance from the original lines except for the male fertile ones.3.Combining ability analysis of nuclear sterile lines:Under two fertilization levels in the greenhouse and the field random group experiment trial,the combining ability of the replacement system and the hybrid species prepared by the original system was basically consistent.indicating that the sterile line was not affected by the sterility gene,and,therefore,can be applied to rice hybrid breeding.In addition,normal fertilization can significantly increase yield.4.Normal fertilization can significantly increase rice production:Under normal fertilization,compared with CK.the number of tillers and single plant yields associated with yield increased by 2.3 and 5.1 g,respectively,to very significant levels.Conclusion:The unit trait of nuclear male sterility is not affected by the sterility mutant gene except for the male sterility,and the combining ability of the transgenic nuclear replacement line is consistent with the original line under different environments.