Cloning Of The Thermo-Sensitive Male Sterility Gene OsTMS18 In Rice And Its Photothermo-Sensitive Mechanis

As one of the three major food crops in the world,rice is the staple food for more than half of the world’s population.Cross-breeding has made an important contribution to the yield of rice,and the research of rice male sterility has laid a foundation for the development of hybrid rice in China.The growing area of the two-line hybrid rice mainly composed of photo-thermo-sensitive sterile lines has reached half of the total growing area of hybrid rice.Compared with the three-line method,two-line hybrid rice has the advantages of not being limited by the restore-protection relationship and relatively free mating,which is more conducive to the utilization of heterosis.As the core of two-line hybrid rice,photo-thermo-sensitive male sterile line has important research value.However,at present,the genetic locus used in two-line hybrid breeding of rice in production is relatively single.Moreover,temperature and light are uncontrollable factors,and the sterility instability of the existing two-line male sterile line under extreme climatic conditions greatly limits its application in production.Therefore,it is of great biological significance to explore new photo-thermo-sensitive genic male sterile genetic resources.In our previous work,the fertility recovery mechanism of photoperiod sensitive male sterility has been revealed in Arabidopsis thaliana.In this study,based on the results of previous studies,we conducted chemical mutagenesis on wild-type Zhonghua 11(ZH11)(Oryza sativa L.subsp.Japonica)by ethyl methyl sulfonate(EMS),and obtained a novel thermosensitive male sterile line ostms18-1 after screening and identification.Under the influence of the occasional low temperature tide in Shanghai in 2021,the high temperature sterility of ostms18-1 mutant was significantly better than that of tms5 mutant with the same background of Zhonghua11.Moreover,when the locus was introduced into different rice backgrounds,the plants of the homozygous mutant locus still showed male sterility,so the locus has potential application in two-line hybrid rice breeding.LOC＿Os10g38050 encoding a glucose-methanol-choline(GMC)oxidoreductase in ostms18-1 mutants have a glycine to serine mutation from the second exon of this gene by bulked segregant analysis(BSA).Complementary experiments showed that the thermo-sensitive phenotype of ostms18-1 was caused by the mutation of this gene.Cell analysis showed that the structure of the second layer of pollen outer wall in ostms18-1mutants was abnormal at high temperature,leading to pollen rupture,but the structure of the second layer of pollen outer wall was thinner but more intact at low temperature.Based on the above results,we proposed that the defective pollen wall is sufficient to protect microspore to develop into functional pollen and restore its fertility under low temperature.Sequence analysis and expression analysis revealed that OsTMS18 has its orthologous gene in Arabidopsis thaliana.The knockout mutant attms18 was fertile under normal conditions(24℃),but its fertility was significantly decreased under high temperature conditions(28℃),also exhibiting thermosensitive traits.Electrophoretic migration assay(EMSA)and in vitro protoplast transformation assay showed that OsTMS18 and At TMS18 genes directly regulated Os MS188 and MS188,the key regulators of pollen wall formation in rice and Arabidopsis thaliana,further indicating that the phenotypes of these genes are directly related to pollen wall.These results revealed the common photo-thermo-sensitive male sterility cytological mechanism in different plants.