MutMap Based Identification And Functional Characterization Of Semi-dwarf OsFBK4 Gene In Rice(Oryza Sativa L.)

Rice is one of the staple food for a large portion of the world’s population.Improvement of rice yield and quality over revolutionary breeding technology is instantly desirable to raise access to nutritious foods at a global level for the ever-increasing population.The study of semi-dwarf genes is of great significance in elucidating the regulatory mechanisms of plant growth and development.More importantly,semi-dwarfism is the most valuable agronomic characteristic in crop improvement programs because it affects lodging resistance and grain yield.Accordingly,the improvement of crop productivity through grain yield and optimal plant architecture selection is an essential factor in the rice breeding program.The widespread exploitation of limited semi-dwarf sources might cause a genetic vul nerability for rice varieties.The identification and development of valuable alternative sources for semi-dwarf genes is,therefore,an essential focus for practical rice breeding.In order to add a new source of semi-dwarf genes to the rice breeding program and to further enhance the genetic and molecular mechanisms underlying rice plant height,a new mutant,RE9586,was identified from ethyl methane sulphonate（EMS）treated Oryza sativa L.indica rice variety ZhongHui8015（ZH8015）.Therefore,the present study aims to identify and characterize the genes responsible for the semi-dwarf phenotype of the RE9586 mutant.The RE9586 plants exhibited reduced plant height than the WT at the maturity stage.Scanning electron microscopic observation results showed that the longitudinal length of the uppermost internode of RE9586 significantly reduced,which is most likely the leading cause of its semi-dwarfing phenotype.The RE9586 mutant had pleiotropic effects on panicle length,tiller numbers,grain yield per plant,and seed setting rate.The chi-square（X²）test met the 3:1recessive single-gene genetic inheritance.A rice novel OsFBK4 gene was isolated by employing a MutMap-based approach that combines the strategies of BSA and NGS from the mutant population.Its single recessive allele functions the regulation of the semi-dwarf phenotype in the RE9586 mutant.The protein sequence of OsFBK4encodes the F-box domain and kelch repeat-containing protein.In RE9586 plants,OsFBK4 showed that a point mutation occurred in the 3^rd exon of LOC＿Os02g11790,resulting in a change in amino acid from Arginine to Glycine that was subsequently confirmed by linkage analysis.The study revealed that the gene was predominantly expressed in the internode.The expressions of GA signaling genes such as SRL1,CIGR,GID1,GID2,D1,and SPY were affected in the RE9586 mutant.OsKAO,OsGA3ox2,and OsGA2ox2 GA biosynthesis genes displayed apparent differences in expression level.The OsFBK4 gene localizes in a plasma membrane.The knockout of OsFBK4 by CRISPR/Cas9 showed a short plant height,while OsFBK4 overexpression significantly increased the plant height.The result showed that RE9586 is a GA insensitive mutant implying that OsFBK4 might be involved in GA signaling providing further insights into the mechanisms underlying the regulation of plant height.