Map-based Cloning And Functional Identification Of The Small Panicle And Grains Gene SPG1 In Rice (Oryza Sativa L.)

Rice is one of the staple foods that people eat daily,the cultivation and production of rice plays an important role in the world.However,in recent years,as the world’s population has grown,we have to further increase rice（Oryza sativa L.）production to meet the world’s food needs.In rice breeding,factors such as plant height,panicle type,grain size and leaves are closely related to rice yield.Plant height affects rice lodging resistance,panicle type and grain size directly determine rice yield,and leaves promote the synthesis of carbohydrates such as starch through photosynthesis to affect yield.Therefore,gene cloning and trait improvement of rice plant height,panicle type,grain size and leaf traits will be helpful for improving rice yield.The mutant material in this study was obtained by EMS（ethylmethane sulfonate）mutagenesis of indica maintainer Xida 1B,the mutant showed small panicle and grains,named spg1（small panicle and grains 1）.Using modern biological technology to carried out researches on gene location,agronomic traits investigation,histology observation,gene expression pattern analysis and gene functional analysis of spg1.Get the following results:1.Investigation of agronomic traits of spg1Comparing the wild-type and spg1 phenotypes at the mature stage,it was found that the spg1 plant height was semi-dwarfed,the number of effective tillers increased,the panicle length became shorter,the grain length and width became smaller,and the thousand-grain weight decreased.However,the number of primary and secondary branches of spg1 increased,so the number of grains per panicle of spg1 increased significantly,and the yield was higher than that of the wild-type.2.Histological analysis of spg1To explore the main reasons for the shorter plant height and smaller grains of spg1,we conducted scanning electron microscopy analysis of wild-type and spg1 sheath cells at the seedling stage and found that the length of spg1 sheath cells became shorter,but the cell width did not change significantly.Analysis of paraffin sections of wild-type and spg1 at the booting stage showed that the length and width of spg1 parenchyma cells became smaller,so the smaller spg1 cells lead to shorter plant height.In addition,we took the wild-type and mutant spikelets at the booting stage for paraffin section and scanning electron microscopy analysis,the results showed that the number of spg1glume cells decreased,which resulted in smaller grains.3.Scanning electron microscope observation of spg1 leavesThe spg1 leaves were smoother than the wild-type.Observing wild-type and spg1leaves at mature stage by scanning electron microscope,we found that the number of the trichomes of spg1 was significantly less than that of wild-type.Therefore,the trichomes number of spg1 decreased lead to the leaf became smoother.4.Genetic analysis and gene mapping of SPG1The hybrid combination of mutant spg1 and wild-type restorer line Jinhui10 was prepared,the phenotypes of the F₁ population were similar to that of the wild-type,however,the F₂ population obtained by selfing the F₁ population has the phenomenon of trait separation.Count all the individual plants of F₂generation,and the chi-square test showed that the segregation ratio complies with the gene segregation law of 3:1,indicating that spg1 is a single-gene mutation.Through map-based cloning,the mutant gene was finally located in the 70kb range between the v-10 and v-9 markers of chromosome 2.There are 9 annotated genes in this interval,by DNA sequencing we found that LOC＿Os02g25230 of spg1 has a deletion of base C at position 953 of exon,which was preliminarily designated as a candidate gene.In order to verify this candidate gene,a complementary vector of the gene was constructed and transformed into spg1mutant plants for complementation experiments.We observing the positive plants at the mature stage found that the phenotypes of the positive plants were restored to the wild-type level.The DNA sequencing results of the positive plants showed that the mutation site sequence of the positive transgenic plants was double peak.Prove that the target gene is LOC＿Os02g25230.5.Subcellular localization of SPG1The entire coding sequence of SPG1 was fused to the N-terminus of green fluorescent protein（GFP）to construct the pAN580-SPG1 vector and transformed into rice protoplasts.The experimental results showed that the SPG1-GFP fusion protein signal coincides with the nuclear Marker signal,indicating that SPG1 is located in the nucleus.6.Expression pattern analysis of SPG1Extracting RNA from various tissues of wild type for semi-quantitative and quantitative PCR experiments,the results showed that SPG1 was expressed in roots,stems,leaves,branches,panicles and other parts.The GUS staining results of each tissue part of the SPG1 gene promoter+GUS positive transgenic plants are consistent with the quantitative and semi-quantitative expression results.All showed that the SPG1was expressed in all tissues of rice,and the highest expression was in the panicles and stems.7.Functional analysis of SPG1Through the phenotype identification of transgenic plants,we found that the interference transgenic plants at the mature stage had the shorter plant height,shorter panicle length,smaller grains,and less trichome numbers.While the number of trichomes of the overexpressing transgenic plants was significantly increased.It shows that SPG1 positively regulates the development of trichomes.